High affinity nucleic acid ligands of complement system proteins

ABSTRACT

Methods are described for the identification and preparation of high-affinity Nucleic Acid Ligands to Complement System Proteins. Methods are described for the identification and preparation of high affinity Nucleic Acid Ligands to Complement System Proteins C1q, C3 and C5. Included in the invention are specific RNA ligands to C1q, C3 and C5 identified by the SELEX method.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 10/037,282, filed Jan. 3, 2002, which is a continuation of U.S.patent application Ser. No. 09/163,025, filed Sep. 29, 1998, now U.S.Pat. No. 6,395,888, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/023,228, filed Feb. 12, 1998, now U.S. Pat. No.6,140,490, which is a continuation-in-part of PCT/US97/01739(International Publication No. WO 97/28178), filed Jan. 30, 1997, whichis a continuation-in-part of U.S. patent. application Ser. No.08/595,335, filed Feb. 1, 1996, now abandoned. This application is alsoa continuation-in-part of U.S. patent application Ser. No. 10/037,986,filed Oct. 18, 2001, which is a continuation of U.S. patent applicationSer. No. 09/502,344, filed Feb. 10, 2000, now U.S. Pat. No. 6,331,398,which is a continuation of U.S. Patent. application Ser. No. 09/143,190,filed Aug. 27, 1998, now U.S. Pat. No. 6,110,900, which is acontinuation of U.S. patent application Ser. No. 08/469,609, filed Jun.6, 1995, now U.S. Pat. No. 5,843,653, which is a continuation of U.S.patent application Ser. No. 08/428,964, filed Apr. 25, 1995, nowabandoned. U.S. patent application Ser. No. 08/469,609 is also acontinuation of U.S. patent application Ser. No. 08/409,442, filed Mar.24, 1995, now U.S. Pat. No. 5,696,249 and a continuation of U.S. patentapplication Ser. No. 08/412,110, filed Mar. 27, 1995, now U.S. Pat. No.5,670,637. Ser. Nos. 08/428,964, 08/409,442, and 08/412,110 arecontinuations of U.S. patent application Ser. No. 07/714,131, filed Jun.10, 1991, now U.S. Pat. No. 5,475,096, which is a continuation-in-partapplication of U.S. patent application Ser. No. 07/536,428, filed Jun.11, 1990, now abandoned.

FIELD OF THE INVENTION

[0002] Described herein are methods for identifying and preparinghigh-affinity Nucleic Acid Ligands to Complement System Proteins. Themethod utilized herein for identifying such Nucleic Acid Ligands iscalled SELEX™, an acronym for Systematic Evolution of Ligands byEXponential enrichment. Described herein are methods for identifying andpreparing high-affinity Nucleic Acid Ligands to the Complement SystemProteins C1q, C3 and C5. This invention includes high affinity NucleicAcid Ligands of C1q, C3 and C5. Also disclosed are RNA ligands of C1q,C3 and C5. Also disclosed are Nucleic Acid Ligands that inhibit and/oractivate the Complement System. The oligonucleotides of the presentinvention are useful as pharmaceuticals or diagnostic agents.

BACKGROUND OF THE INVENTION

[0003] The complement system comprises a set of at least 20 plasma andmembrane proteins that act together in a regulated cascade system toattack extracellular forms of pathogens (Janeway et al. (1994)Immunobiology: The Immune System in Health and Disease. Current BiologyLtd, San Francisco, pp. 8:35-8:55; Morgan (1995) Crit. Rev. in Clin Lab.Sci. 32(3):265-298). There are two distinct enzymatic activationcascades, the classical and alternative pathways, and a non-enzymaticpathway known as the membrane attack pathway.

[0004] The classical pathway is usually triggered by an antibody boundto a foreign particle. It comprises several components, C1, C4, C2, C3and C5 (listed by order in the pathway). Initiation of the classicalpathway of the Complement System occurs following binding and activationof the first complement component (C1) by both immune and non-immuneactivators (Cooper (1985) Adv. Immunol. 37:151). C1 comprises acalcium-dependent complex of components C1q, C1r and C1s, and isactivated through binding of the C1q component. C1 q contains sixidentical subunits and each subunit comprises three chains (the A, B andC chains). Each chain has a globular head region which is connected to acollagen-like tail. Binding and activation of C1q by antigen-antibodycomplexes occurs through the C1q head group region. Numerousnon-antibody C1q activators, including proteins, lipids and nucleicacids (Reid et al. (1993) The Natural Immune System: Humoral Factors. E.Sim, ed. IRL Press, Oxford, p. 151) bind and activate through a distinctsite on the collagen-like stalk region.

[0005] Non-antibody C1q protein activators include C-reactive protein(CRP) (Jiang et al. (1991) J. Immunol. 146:2324) and serum amyloidprotein (SAP) (Bristow et al. (1986) Mol. Immunol. 23:1045); these willactivate C1q when aggregated by binding to phospholipid or carbohydrate,respectively. Monomeric CRP or SAP do not activate C1q. C1q is alsoactivated through binding to aggregated β-amyloid peptide (Schultz etal. (1994) Neurosci. Lett. 175:99; Snyder et al. (1994) Exp. Neurol.128:136), a component of plaques seen in Alzheimer's disease (Jiang etal. (1994) J. Immunol. 152:5050; Eikelenboom and Stam (1982) ActaNeuropathol (Berl) 57:239; Eikelenboom et al. (1989) Virchows Arch. [B]56:259; Rogers et al. (1992) Proc. Natl. Acad. Sci. USA 89:10016;Dietzschold et al. (1995) J. Neurol. Sci. 130:11). C1q activation mightalso exacerbate the tissue damage associated with Alzheimer's disease.These activators bind C1q on its collagen-like region, distant from thehead-group region where immunoglobulin activators bind. Other proteinswhich bind the C1q collagen-like region include collagen (Menzel et al.(1981) Biochim. Biophys. Acta 670:265), fibronectin (Reid et al. (1984)Acta Pathol. Microbiol. Immunol. Scand. Sect. C 92 (Suppl. 284):11),laminin (Bohnsack et al. (1985) Proc. Natl. Acad. Sci. USA 82:3824),fibrinogen and fibrin (Entwistle et al. (1988) Biochem. 27:507), HIVrsgp41 (Stoiber et al. (1995) Mol. Immunol. 32:371), actin (Nishioka etal. (1982) Biochem. Biophys. Res. Commun. 108:1307) and tobaccoglycoprotein (Koethe et al. (1995) J. Immunol. 155:826).

[0006] C1q also binds and can be activated by anionic carbohydrates(Hughes-Jones et al. (1978) Immunology 34:459) includingmucopolysaccharides (Almeda et al. (1983) J. Biol. Chem. 258:785),fucans (Blondin et al. (1994) Mol. Immunol. 31 :247), proteoglycans(Silvestri et al. (1981) J. Biol. Chem. 256:7383), and by lipidsincluding lipopolysaccharide (LPS) (Zohair et al. (1989) Biochem. J.257:865; Stoiber et al. (1994) Eur. J. Immunol. 24:294). Both DNA(Schravendijk and Dwek (1982) Mol. Immunol. 19:1179; Rosenberg et al.(1988) J. Rheumatol 15:1091; Uwatoko et al. (1990) J. Immunol. 144:3484)and RNA (Acton et al. (1993) J. Biol. Chem. 268:3530) can also bind andpotentially activate C1q. Intracellular components which activate C1qinclude cellular and subcellular membranes (Linder (1981) J. Immunol.126:648; Pinckard et al. (1973) J. Immunol. 110: 1376; Storrs et al.(1981) J. Biol. Chem. 256:10924; Giclas et al. (1979) J. Immmunol.122:146; Storrs et al. (1983) J. Immunol. 131:416), intermediatefilaments (Linder et al. (1979) Nature 278:176) and actin (Nishioka etal. (1982) Biochem. Biophys. Res. Commun. 108:1307). All of theseinteractions would recruit the classical pathway for protection againstbacterial (or viral) infection, or as a response to tissue injury (Li etal. (1994) J. Immunol. 152:2995) in the absence of antibody.

[0007] A binding site for non-antibody activators including CRP (Jianget al. (1991) J. Immunol. 146:2324), SAP (Ying et al. (1993) J. Immunol.150:169), β-amyloid peptide (Newman (1994) Curr. Biol. 4:462) and DNA(Jiang et al. (1992) J. Biol. Chem. 267:25597) has been localized to theamino terminus of C1q A chain at residues 14-26. A synthetic peptidecomprising this sequence effectively inhibits both binding andactivation. The peptide 14-26 contains several basic residues andmatches one of the heparin binding motifs (Yabkowitz et al. (1989) J.Biol. Chem. 264:10888; Cardin et al. (1989) Arteriosclerosis 9:21). Thepeptide is also highly homologous with peptide 145-156 incollagen-tailed acetylcholinesterase; this site is associated withheparin-sulfate basement membrane binding (Deprez et al. (1995) J. Biol.Chem. 270:11043). A second C1q A chain site at residues 76-92 also mightbe involved in weaker binding; this site is at the junction of theglobular head region and the collagen-like tail.

[0008] The second enzymatically activated cascade, known as thealternative pathway, is a rapid, antibody-independent route for theComplement System activation and amplification. The alternative pathwaycomprises several components, C3, Factor B, and Factor D. Activation ofthe alternative pathway occurs when C3b, a proteolytic cleavage form ofC3, is bound to an activating surface such as a bacterium. Factor B isthen bound to C3b, and cleaved by Factor D to yield the active enzyme,Ba. The enzyme Ba then cleaves more C3 to C3b, producing extensivedeposition of C3b-Ba complexes on the activating surface. When a secondC3b is deposited, forming a C3b-C3b-Ba complex, the enzyme can thencleave C5 and trigger activation of the terminal pathway.

[0009] The non-enzymatic terminal pathway, also known as the membraneattack pathway, comprises the components C5, C6, C7, C8 and C9.Activation of this membrane attack pathway results when the C5 componentis enzymatically cleaved by either the classical or alternative pathwayto yield the small C5a polypeptide (9 kDa) and the large C5b fragment(200 kDa). The C5a polypeptide binds to a 7 transmembrane G-proteincoupled receptor which was originally described on leukocytes and is nowknown to be expressed on a variety of tissues including hepatocytes(Haviland et al. (1995) J. Immunol. 154:1861) and neurons (Gasque et al.(1997) Am. J. Pathol. 150:31). The C5a molecule is the primarychemotactic component of the human Complement System and can trigger avariety of biological responses including leukocyte chemotaxis, smoothmuscle contraction, activation of intracellular signal transductionpathways, neutrophil-endothelial adhesion (Mulligan et al. (1997) J.Immunol. 158:1857), cytokine and lipid mediator release and oxidantformation. The larger C5b fragment binds sequentially to latercomponents to form the C5b-9 membrane attack complex (MAC). The C5b-9MAC can directly lyse erythrocytes, and in greater quantities is lyticfor leukocytes and is damaging to tissues such as muscle, epithelial andendothelial cells (Stahl et al. (1997) Circ. Res. 76:575). In sublyticamounts the MAC can stimulate upregulation of adhesion molecules,intracellular calcium increase and cytokine release (Ward (1996) Am. J.Pathol. 149:1079). In addition, the C5b-9 MAC can stimulate cells suchas endothelial cells and platelets without causing cell lysis. Thenon-lytic effects of C5a and the C5b-9 MAC are sometimes quite similar.

[0010] The Complement System has an important role in defense againstbacterial and viral infection, and possibly in immune surveillanceagainst tumors. This is demonstrated most clearly in humans who aredeficient in complement components. Individuals deficient in earlycomponents (C1, C4, C2 or C3) suffer from recurrent infections, whileindividuals deficient in late components (C5 through C9) are susceptibleto nisseria infection. Complement classical pathway is activated onbacteria by antibodies, by binding of CRP or SAP, or by directactivation through LPS. Complement alternative pathway is activatedthrough binding of C3 to the cell coat. Complement can be activated byviruses through antibodies, and can also be activated on viral infectedcells because these are recognized as foreign. In a similar way,transformed cells can be recognized as foreign and can be lysed by theComplement System or targeted for immune clearance.

[0011] Activation of the Complement System can and has been used fortherapeutic purposes. Antibodies which were produced against tumor cellswere then used to activate the Complement System and cause tumorrejection. Also, the Complement System is used together with polyclonalor monoclonal antibodies to eliminate unwanted lymphocytes. For example,anti-lymphocyte globulin or monoclonal anti-T-cell antibodies are usedprior to organ transplantation to eliminate lymphocytes which wouldotherwise mediate rejection.

[0012] Although the Complement System has an important role in themaintenance of health, it has the potential to cause or contribute todisease. The Complement System has been implicated in numerous renal,rheumatological, neurological, dermatological, hematological,vascular/pulmonary, allergy, infectious, biocompatibility/shock andother diseases or conditions (Morgan (1995) Crit. Rev. in Clin Lab. Sci.32(3 :265-298; Matis and Rollins (1995) Nature Medicine 1(8):839-842).The Complement System is not necessarily the only cause of the diseasestate, but it may be one of several factors, each of which contributesto pathogenesis.

[0013] Several pharmaceuticals have been developed that inhibit theComplement System in vivo, however, many cause toxicity or are poorinhibitors (Morgan (1995) Crit. Rev. in Clin Lab. Sci. 32(3):265-298).Heparins, K76COOH and nafamstat mesilate have been shown to be effectivein animal studies (Morgan (1995) Crit. Rev. in Clin Lab. Sci.32(3):265-298). Recombinant forms of naturally occurring inhibitors ofthe Complement System have been developed or are under consideration,and these include the membrane regulatory proteins Complement Receptor 1(CR1), Decay Accelerating Factor (DAF), Membrane Cofactor Protein (MCP)and CD59.

[0014] C5 is an attractive target for the development of a ComplementSystem inhibitor, as both the classical and alternative pathwaysconverge at component C5 (Matis and Rollins (1995) Nature Medicine1(8):839-842). In addition, inhibition of C5 cleavage blocks both theC5a and the C5b effects on leukocytes and on tissue such as endothelialcells (Ward (1996) Am. J. Pathol. 149:1079); thus C5 inhibition can havetherapeutic benefits in a variety of diseases and situations, includinglung inflammation (Mulligan et al. (1998) J. Clin. Invest. 98:503),extracorporeal complement activation (Rinder et al. (1995) J. Clin.Invest. 96:1564) or antibody-mediated complement activation (Bieseckeret al. (1989) J. Immunol. 142:2654). Matis and Rollins ((1995) NatureMedicine 1(8):839-842) have developed C5-specific monoclonal antibodiesas an anti-inflammatory biopharmaceutical. Both C5a and the MAC havebeen implicated in acute and chronic inflammation associated with humandisease, and their role in disease states has been confirmed in animalmodels. C5a is required for complement- and neutrophil-dependent lungvascular injury (Ward (1997) J. Lab. Clin. Med. 129:400; Mulligan et al.(1998) J. Clin. Invest. 98:503), and is associated with neutrophil andplatelet activation in shock and in burn injury (Schmid et al. (1997)Shock 8:119). The MAC mediates muscle injury in acute autoimmunemyasthenia gravis (Biesecker and Gomez (1989) J. Immunol. 142:2654),organ rejection in transplantation (Baldwin et al. (1995)Transplantation 59:797; Brauer et al. (1995) Transplantation 59:288;Takahashi et al. (1997) Immunol. Res. 16:273) and renal injury inautoimmune glomerulonephritis (Biesecker (1981) J. Exp. Med. 39:1779;Nangaku (1997) Kidney Int. 52:1570). Both C5a and the MAC are implicatedin acute myocardial ischemia (Homeister and Lucchesi (1994) Annu. Rev.Pharmacol. Toxicol. 34:17), acute (Bednar et al. (1997) J. Neurosurg.86:139) and chronic CNS injury (Morgan (1997) Exp. Clin. Immunogenet.14:19), leukocyte activation during extracorporeal circulation (Sun etal. (1995) Nucleic Acids Res. 23:2909; Spycher and Nydegger (1995)Infushionsther. Transfusionsmed. 22:36) and in tissue injury associatedwith autoimmune diseases including arthritis and lupus (Wang et al.(1996) Immunology 93:8563). Thus, inhibiting cleavage of C5 preventsgeneration of two potentially damaging activities of the ComplementSystem. Inhibiting C5a release eliminates the major Complement Systemchemotactic and vasoactive activity, and inhibiting C5b formation blocksassembly of the cytolytic C5b-9 MAC. Furthermore, inhibition of C5prevents injury by the Complement System while leaving intact importantComplement System defense and clearance mechanisms, such as C3 and C1qphagocytic activity, clearance of immune complexes and the innate immuneresponse (Carrol (1998) Ann. Rev. Immunol. 16:545).

[0015] C3 is an attractive target for the development of a ComplementSystem inhibitor, as it is common to both pathways. Inhibition of C3using recombinant versions of a natural inhibitors (Kalli et al. (1994)Springer Semin. Immunopathol. 15:417) can prevent cell-mediated tissueinjury (Mulligan et al. (1992) J. Immunol. 148:1479) and this has beenshown to have therapeutic benefit in diseases such as myocardialinfarction (Weisman et al. (1990) Science 249:146) and liverischemia/reperfusion (Chavez-Cartaya et al. (1995) Transplantation59:1047). Controlling C3 limits most biological activities of theComplement System. Most natural inhibitors, including DAF, MCP, CR1 andFactor H target C3.

[0016] SELEX™

[0017] A method for the in vitro evolution of Nucleic Acid moleculeswith highly specific binding to target molecules has been developed.This method, Systematic Evolution of Ligands by EXponential enrichment,termed the SELEX process, is described in U.S. patent application Ser.No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution ofLigands by Exponential Enrichment,” now abandoned; U.S. patentapplication Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “NucleicAcid Ligands,” now U.S. Pat. No. 5,475,096; U.S. patent application Ser.No. 07/931,473, filed Aug. 17, 1992, entitled “Methods for IdentifyingNucleic Acid Ligands,” now U.S. Pat. No. 5,270,163 (see also WO91/19813), each of which is herein specifically incorporated byreference in its entirety. Each of these applications, collectivelyreferred to herein as the SELEX Patent Applications, describes afundamentally novel method for making a Nucleic Acid Ligand to anydesired Target molecule.

[0018] The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of Nucleic Acids, preferably comprising asegment of randomized sequence, the SELEX method includes steps ofcontacting the mixture with the Target under conditions favorable forbinding, partitioning unbound Nucleic Acids from those Nucleic Acidswhich have bound specifically to Target molecules, dissociating theNucleic Acid-Target complexes, amplifying the Nucleic Acids dissociatedfrom the Nucleic Acid-Target complexes to yield a ligand-enrichedmixture of Nucleic Acids, then reiterating the steps of binding,partitioning, dissociating and amplifying through as many cycles asdesired to yield highly specific, high affinity Nucleic Acid Ligands tothe Target molecule.

[0019] The basic SELEX method has been modified to achieve a number ofspecific objectives. For example, U.S. patent application Ser. No.07/960,093, filed Oct. 14, 1992, entitled “Method for Selecting NucleicAcids on the Basis of Structure,” now abandoned (see also U.S. Pat. No.5,707,796), describes the use of the SELEX method in conjunction withgel electrophoresis to select Nucleic Acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” now abandoned, (see also U.S. Pat. No. 5,763,177)describes a SELEX-based method for selecting Nucleic Acid Ligandscontaining photoreactive groups capable of binding and/orphotocrosslinking to and/or photoinactivating a Target molecule. U.S.patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled“High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” now abandoned (see also U.S. Pat. No.5,580,737), describes a method for identifying highly specific NucleicAcid Ligands able to discriminate between closely related molecules,termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filedOct. 25, 1993, entitled “Systematic Evolution of Ligands by EXponentialEnrichment: Solution SELEX,” now abandoned, (see also U.S. Pat. No.5,567,588) and U.S. patent application Ser. No. 08/792,075, filed Jan.31, 1997, entitled “Flow Cell SELEX,” now U.S. Pat. No. 5,861,254,describe SELEX-based methods which achieve highly efficient partitioningbetween oligonucleotides having high and low affinity for a Targetmolecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21,1992, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” now U.S.Pat. No. 5,496,938, describes methods for obtaining improved NucleicAcid Ligands after the SELEX process has been performed. U.S. patentapplication Ser. No. 08/400,440, filed Mar. 8, 1995, entitled“Systematic Evolution of Ligands by EXponential Enrichment:Chemi-SELEX,” now U.S. Pat. No. 5,705,337, describes methods forcovalently linking a ligand to its Target.

[0020] The SELEX method encompasses the identification of high-affinityNucleic Acid Ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified Nucleic Acid Ligands containingmodified nucleotides are described in U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, entitled “High Affinity Nucleic AcidLigands Containing Modified Nucleotides,” now abandoned, (see also U.S.Pat. No. 5,660,985) that describes oligonucleotides containingnucleotide derivatives chemically modified at the 5- and 2′-positions ofpyrimidines. U.S. patent application Ser. No. 08/134,028, now U.S. Pat.No. 5,580,737, supra, describes highly specific Nucleic Acid Ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” now abandoned, describes oligonucleotidescontaining various 2′-modified pyrimidines.

[0021] The SELEX method encompasses combining selected oligonucleotideswith other selected oligonucleotides and non-oligonucleotide functionalunits as described in U.S. patent application Ser. No. 08/284,063, filedAug. 2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX,” now U.S. Pat. No. 5,637,459 and U.S. patentapplication Ser. No. 08/234,997, filed Apr. 28, 1994, entitled“Systematic Evolution of Ligands by Exponential Enrichment: BlendedSELEX,” now U.S. Pat. No. 5,683,867, respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules. Eachof the above described patent applications which describe modificationsof the basic SELEX procedure are specifically incorporated by referenceherein in their entirety.

BRIEF SUMMARY OF THE INVENTION

[0022] The present invention includes methods of identifying andproducing Nucleic Acid Ligands to Complement System Proteins andhomologous proteins and the Nucleic Acid Ligands so identified andproduced. By homologous proteins it is meant a degree of amino acidsequence identity of 80% or more. Exemplified herein is a method ofidentifying and producing Nucleic Acid Ligands to C1q, C3 and C5, andthe Nucleic Acid Ligands so produced. Nucleic Acid Ligand sequences areprovided that are capable of binding specifically to C1q, C3 and C5. Inparticular, RNA sequences are provided that are capable of bindingspecifically the C1q, C3 and C5. Specifically included in the inventionare the RNA ligand sequences shown in Tables 2-6, 8, 10 and 12-13 andFIGS. 5A-B (SEQ ID NOS: 5-155 and 160-196). Also included in theinvention are Nucleic Acid Ligands that inhibit the function of proteinsof the Complement System. Specifically included in the invention hereinare RNA ligands that inhibit the function of C1 q, C3 and C5. Alsoincluded are Nucleic Acid Ligands that inhibit and/or activate theComplement System.

[0023] Further included in this invention is a method of identifyingNucleic Acid Ligands and Nucleic Acid Ligand sequences to ComplementSystem Proteins comprising the steps of (a) preparing a CandidateMixture of Nucleic Acids, (b) contacting the Candidate Mixture ofNucleic Acids with a Complement System Protein, (c) partitioning betweenmembers of said Candidate Mixture on the basis of affinity to saidComplement System Protein, and (d) amplifying the selected molecules toyield a mixture of Nucleic Acids enriched for Nucleic Acid sequenceswith a relatively higher affinity for binding to said Complement SystemProtein.

[0024] Also included in this invention is a method of identifyingNucleic Acid Ligands and Nucleic Acid Ligand sequences to C1q, C3 andC5, comprising the steps of (a) preparing a Candidate Mixture of NucleicAcids, (b) contacting the Candidate Mixture of Nucleic Acids with C1q,C3 or C5, (c) partitioning between members of said Candidate Mixture onthe basis of affinity to C1q, C3 or C5, and (d) amplifying the selectedmolecules to yield a mixture of Nucleic Acids enriched for Nucleic Acidsequences with a relatively higher affinity for binding to C1q, C3 orC5.

[0025] More specifically, the present invention includes the RNA ligandsto C1q, C3 and C5 identified according to the above-described method,including RNA ligands to C1q, including those ligands shown in Table 2(SEQ ID NOS:5-20) and Table 6 (SEQ ID NOS: 84-155), RNA ligands to C3,including those sequences shown in Table 3 (SEQ ID NOS:21-46), and RNAligands to C5, including those sequences shown in Table 4 (SEQ IDNOS:47-74), Table 5 (SEQ ID NOS:76-83), Table 8 (SEQ ID NOS:75,160-162), Table 10 (SEQ ID NOS:163-189), Table 12 (SEQ ID NOS:190-192),Table 13 (SEQ ID NOS:194-196) and FIGS. 5A-B (SEQ ID NOS:160 and 193).Also included are RNA ligands to C1q, C3 and C5 that are substantiallyhomologous to any of the given ligands and that have substantially thesame ability to bind C1q, C3 or C5, and inhibit the function of C1q, C3or C5. Further included in this invention are Nucleic Acid Ligands toC1q, C3 and C5 that have substantially the same structural form as theligands presented herein and that have substantially the same ability tobind C1q, C3 or C5 and inhibit the function of C1q, C3 or C5.

[0026] The present invention also includes modified nucleotide sequencesbased on the RNA ligands identified herein and mixtures of the same.

BRIEF DESCRIPTION OF THE FIGURES

[0027]FIG. 1 shows the results of an inhibition assay in which 2′-F RNAligands C 12 (SEQ ID NO:59), A6 (SEQ ID NO:48), K7 (SEQ ID NO:50), C9(SEQ ID NO:58), E5c (SEQ ID NO:47) and F8 (SEQ ID NO:49) to human C5were incubated with antibody-coated sheep erythrocytes and whole humanserum. The results are presented as optical density (OD) versusconcentration of ligand in nM.

[0028]FIG. 2 shows the % C5a generation as a function of concentrationof clone C6 (SEQ ID NO:51).

[0029]FIG. 3A shows a sequencing gel of 5′-kinase-labeled clone C6 (SEQID NO:51) after alkaline hydrolysis or digestion with T₁ nuclease. The3′-sequence (5′-end labeled) is aligned with the alkaline hydrolysisladder. On the left is the T₁ ladder and on the right are RNA selectedwith 5× and 1× concentrations of C5. The boundary where removal of abase eliminates binding is shown by the arrow. The asterisk shows a Gwhich is hypersensitive to T₁.

[0030]FIG. 3B shows a sequencing gel of 3′-pCp-ligated clone C6 afteralkaline hydrolysis or digestion with T₁ nuclease. The 5′-sequence(3′-end-labled) is aligned with the alkaline hydrolysis ladder. The T₁and protein lanes, boundary and hypersensitive G nucleotides are asdescribed for FIG. 3A.

[0031]FIG. 4 shows the results of the 2′-O-methyl interference assay.Positions where 2′-OH purines can be substituted with 2′-O-methyl weredetermined from binding interference. Plotted is the ratio of (theintensity of bands selected by protein)/(the band intensity foroligonucleotides not selected by protein) with a linear curve fit (opencircles). The same ratio for mixed 2′-OH:2′-OMe nucleotides is alsoplotted (closed circles).

[0032]FIG. 5A shows the proposed structure of the 38 mer truncate (SEQID NO:160) of clone C6 (SEQ ID NO:51) together with alternative bases.

[0033]FIG. 5B shows the 2′-O-methyl substitution pattern of a 38 mertruncate (SEQ ID NO: 193 of clone C6 (SEQ ID NO:51). Positions where2′-OMe substitutions can be made are shown in bold. Positions which mustbe 2′-OH are underlined.

[0034]FIG. 6 shows the % hemolysis verses concentration of nucleic acidligand (μm) for a 38 mer truncate of clone YL-13 (SEQ ID NO: 175)without 2′-OMe substitution (SEQ ID NO:194; open circles), with a 2′-OMesubstitution at position 20 (SEQ ID NO:195; closed triangles) and with2′-OMe substitutions at positions 2, 7, 8, 13, 14, 15, 20, 21, 22, 26,27, 28, 36 and 38 (SEQ ID NO:196; closed circles).

DETAILED DESCRIPTION OF THE INVENTION

[0035] This application describes Nucleic Acid Ligands to ComplementSystem Proteins identified generally according to the method known asSELEX. As stated earlier, the SELEX technology is described in detail inthe SELEX Patent Applications which are incorporated herein by referencein their entirety. Certain terms used to describe the invention hereinare defined as follows:

[0036] “Nucleic Acid Ligand” as used herein is a non-naturally occurringNucleic Acid having a desirable action on a Target. A desirable actionincludes, but is not limited to, binding of the Target, catalyticallychanging the Target, reacting with the Target in a way whichmodifies/alters the Target or the functional activity of the Target,covalently attaching to the Target as in a suicide inhibitor, andfacilitating the reaction between the Target and another molecule. Inthe preferred embodiment, the desirable action is specific binding to aTarget molecule, such Target molecule being a three dimensional chemicalstructure other than a polynucleotide that binds to the Nucleic AcidLigand through a mechanism which predominantly depends on Watson/Crickbase pairing or triple helix binding, wherein the Nucleic Acid Ligand isnot a Nucleic Acid having the known physiological function of beingbound by the Target molecule. Nucleic Acid Ligands include Nucleic Acidsthat are identified from a Candidate Mixture of Nucleic Acids, saidNucleic Acid Ligand being a ligand of a given Target by the methodcomprising: a) contacting the Candidate Mixture with the Target, whereinNucleic Acids having an increased affinity to the Target relative to theCandidate Mixture may be partitioned from the remainder of the CandidateMixture; b) partitioning the increased affinity Nucleic Acids from theremainder of the Candidate Mixture; and c) amplifying the increasedaffinity Nucleic Acids to yield a ligand-enriched mixture of NucleicAcids.

[0037] “Candidate Mixture” is a mixture of Nucleic Acids of differingsequence from which to select a desired ligand. The source of aCandidate Mixture can be from naturally-occurring Nucleic Acids orfragments thereof, chemically synthesized Nucleic Acids, enzymaticallysynthesized Nucleic Acids or Nucleic Acids made by a combination of theforegoing techniques. In a preferred embodiment, each Nucleic Acid hasfixed sequences surrounding a randomized region to facilitate theamplification process.

[0038] “Nucleic Acid” means both DNA, RNA, single-stranded ordouble-stranded and any chemical modifications thereof. Modificationsinclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability, hydrogenbonding, electrostatic interaction, and fluxionality to the Nucleic AcidLigand bases or to the Nucleic Acid Ligand as a whole. Suchmodifications include, but are not limited to, 2′-position sugarmodifications, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbonemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping.

[0039] “SELEX™” methodology involves the combination of selection ofNucleic Acid Ligands which interact with a Target in a desirable manner,for example binding to a protein, with amplification of those selectedNucleic Acids. Iterative cycling of the selection/amplification stepsallows selection of one or a small number of Nucleic Acids whichinteract most strongly with the Target from a pool which contains a verylarge number of Nucleic Acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. In the presentinvention, the SELEX methodology is employed to obtain Nucleic AcidLigands to C1q, C3 and C5. The SELEX methodology is described in theSELEX Patent Applications.

[0040] “Target” means any compound or molecule of interest for which aligand is desired. A Target can be a protein, peptide, carbohydrate,polysaccharide, glycoprotein, hormone, receptor, antigen, antibody,virus, substrate, metabolite, transition state analog, cofactor,inhibitor, drug, dye, nutrient, growth factor, etc. without limitation.In this application, the Target is a Complement System Protein,preferably C1q, C3 and C5.

[0041] “Complement System Protein” means any protein or component of theComplement System including, but not limited to, C1, C1q, C1r, C1s, C2,C3, C3a, C3b, C4, C4a, C5, C5a, C5b, C6, C7, C8, C9, Factor B (B),Factor D (D), Factor H (H) and receptors thereof, and other soluble andmembrane inhibitors/control proteins.

[0042] “Complement System” is a set of plasma and membrane proteins thatact together in a regulated cascade system to attack extracellular formsof pathogens or infected or transformed cells, and in clearance ofimmune reactants or cellular debris. The Complement System can beactivated spontaneously on certain pathogens or by antibody binding tothe pathogen. The pathogen becomes coated with Complement SystemProteins (opsonized) for uptake and destruction. The pathogen can alsobe directly lysed and killed. Similar mechanisms target infected,transformed or damaged cells. The Complement System also participates inclearance of immune and cellular debris.

[0043] The SELEX process is described in U.S. patent application Ser.No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution ofLigands by EXponential Enrichment,” now abandoned; U.S. patentapplication Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “NucleicAcid Ligands,” now U.S. Pat. No. 5,475,096; U.S. patent application Ser.No. 07/931,473, filed Aug. 17, 1992, entitled “Methods for IdentifyingNucleic Acid Ligands,” now U.S. Pat. No. 5,270,163 (see also WO91/19813). These applications, each specifically incorporated herein byreference, are collectively called the SELEX Patent Applications.

[0044] In its most basic form, the SELEX process may be defined by thefollowing series of steps:

[0045] 1) A Candidate Mixture of Nucleic Acids of differing sequence isprepared. The Candidate Mixture generally includes regions of fixedsequences (i.e., each of the members of the Candidate Mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the Target, or (c) to enhance the concentration of agiven structural arrangement of the Nucleic Acids in the CandidateMixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

[0046] 2) The Candidate Mixture is contacted with the selected Targetunder conditions favorable for binding between the Target and members ofthe Candidate Mixture. Under these circumstances, the interactionbetween the Target and the Nucleic Acids of the Candidate Mixture can beconsidered as forming Nucleic Acid-Target pairs between the Target andthose Nucleic Acids having the strongest affinity for the Target.

[0047] 3) The Nucleic Acids with the highest affinity for the Target arepartitioned from those Nucleic Acids with lesser affinity to the Target.Because only an extremely small number of sequences (and possibly onlyone molecule of Nucleic Acid) corresponding to the highest affinityNucleic Acids exist in the Candidate Mixture, it is generally desirableto set the partitioning criteria so that a significant amount of theNucleic Acids in the Candidate Mixture (approximately 5-50%) areretained during partitioning.

[0048] 4) Those Nucleic Acids selected during partitioning as having therelatively higher affinity to the Target are then amplified to create anew Candidate Mixture that is enriched in Nucleic Acids having arelatively higher affinity for the Target.

[0049] 5) By repeating the partitioning and amplifying steps above, thenewly formed Candidate Mixture contains fewer and fewer weakly bindingsequences, and the average degree of affinity of the Nucleic Acids tothe Target will generally increase. Taken to its extreme, the SELEXprocess will yield a Candidate Mixture containing one or a small numberof unique Nucleic Acids representing those Nucleic Acids from theoriginal Candidate Mixture having the highest affinity to the Targetmolecule.

[0050] The SELEX Patent Applications describe and elaborate on thisprocess in great detail. Included are Targets that can be used in theprocess; methods for partitioning Nucleic Acids within a CandidateMixture; and methods for amplifying partitioned Nucleic Acids togenerate enriched Candidate Mixture. The SELEX Patent Applications alsodescribe ligands obtained to a number of target species, including bothprotein Targets where the protein is and is not a Nucleic Acid bindingprotein.

[0051] The SELEX method further encompasses combining selected NucleicAcid Ligands with lipophilic or Non-Immunogenic, High Molecular Weightcompounds in a diagnostic or therapeutic complex as described in U.S.patent application Ser. No. 08/434,465, filed May 4, 1995, entitled“Nucleic Acid Ligand Complexes,” now U.S. Pat. No. 6,011,020. VEGFNucleic Acid Ligands that are associated with a Lipophilic Compound,such as diacyl glycerol or dialkyl glycerol, in a diagnostic ortherapeutic complex are described in U.S. patent application Ser. No.08/739,109, filed Oct. 25, 1996, entitled “Vascular Endothelial GrowthFactor (VEGF) Nucleic Acid Ligand Complexes,” now U.S. Pat. No.5,859,228. VEGF Nucleic Acid Ligands that are associated with aLipophilic Compound, such as a glycerol lipid, or a Non-Immunogenic,High Molecular Weight Compound, such as polyalkylene glycol, are furtherdescribed in U.S. patent application Ser. No. 08/897,351, filed Jul. 21,1997, entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic AcidLigand Complexes,” now U.S. Pat. No. 6,051,698. VEGF Nucleic AcidLigands that are associated with a Non-Immunogenic, High MolecularWeight compound or a lipophilic compound are also further described inPCT/US97/18944, filed Oct. 17, 1997, entitled “Vascular EndothelialGrowth Factor (VEGF) Nucleic Acid Ligand Complexes.” Each of the abovedescribed patent applications which describe modifications of the basicSELEX procedure are specifically incorporated by reference herein intheir entirety.

[0052] Certain embodiments of the present invention provide a complexcomprising one or more Nucleic Acid Ligands to a Complement SystemProtein covalently linked with a Non-Immunogenic, High Molecular Weightcompound or lipophilic compound. A complex as used herein describes themolecular entity formed by the covalent linking of the Nucleic AcidLigand of a Complement System Protein to a Non-Immunogenic, HighMolecular Weight compound. A Non-Immunogenic, High Molecular Weightcompound is a compound between approximately 100 Da to 1,000,000 Da,more preferably approximately 1000 Da to 500,000 Da, and most preferablyapproximately 1000 Da to 200,000 Da, that typically does not generate animmunogenic response. For the purposes of this invention, an immunogenicresponse is one that causes the organism to make antibody proteins. Inone preferred embodiment of the invention, the Non-Immunogenic, HighMolecular Weight compound is a polyalkylene glycol. In the mostpreferred embodiment, the polyalkylene glycol is polyethylene glycol(PEG). More preferably, the PEG has a molecular weight of about 10-80K.Most preferably, the PEG has a molecular weight of about 20-45K. Incertain embodiments of the invention, the Non-Immunogenic, HighMolecular Weight compound can also be a Nucleic Acid Ligand.

[0053] Another embodiment of the invention is directed to complexescomprised of a Nucleic Acid Ligand to a Complement System Protein and alipophilic compound. Lipophilic compounds are compounds that have thepropensity to associate with or partition into lipid and/or othermaterials or phases with low dielectric constants, including structuresthat are comprised substantially of lipophilic components. Lipophiliccompounds include lipids as well as non-lipid containing compounds thathave the propensity to associate with lipids (and/or other materials orphases with low dielectric constants). Cholesterol, phospholipid, andglycerol lipids, such as dialkyl glycerol, diacyl glycerol, and glycerolamide lipids are further examples of lipophilic compounds. In apreferred embodiment, the lipophilic compound is a glycerol lipid.

[0054] The Non-Immunogenic, High Molecular Weight compound or lipophiliccompound may be covalently bound to a variety of positions on theNucleic Acid Ligand to a Complement System Protein, such as to anexocyclic amino group on the base, the 5-position of a pyrimidinenucleotide, the 8-position of a purine nucleotide, the hydroxyl group ofthe phosphate, or a hydroxyl group or other group at the 5′ or 3′terminus of the Nucleic Acid Ligand to a Complement System Protein. Inembodiments where the lipophilic compound is a glycerol lipid, or theNon-Immunogenic, High Molecular Weight compound is polyalkylene glycolor polyethylene glycol, preferably the Non-Immunogenic, High MolecularWeight compound is bonded to the 5′ or 3′ hydroxyl of the phosphategroup thereof. In the most preferred embodiment, the lipophilic compoundor Non-Immunogenic, High Molecular Weight compound is bonded to the 5′hydroxyl of the phosphate group of the Nucleic Acid Ligand. Attachmentof the Non-Immunogenic, High Molecular Weight compound or lipophiliccompound to the Nucleic Acid Ligand of the Complement System Protein canbe done directly or with the utilization of linkers or spacers.

[0055] A linker is a molecular entity that connects two or moremolecular entities through covalent bonds or non-covalent interactions,and can allow spatial separation of the molecular entities in a mannerthat preserves the functional properties of one or more of the molecularentities. A linker can also be referred to as a spacer.

[0056] The complex comprising a Nucleic Acid Ligand to a ComplementSystem Protein and a Non-Immunogenic, High Molecular Weight compound orlipophilic compound can be further associated with a lipid construct.Lipid constructs are structures containing lipids, phospholipids, orderivatives thereof comprising a variety of different structuralarrangements which lipids are known to adopt in aqueous suspension.These structures include, but are not limited to, lipid bilayervesicles, micelles, liposomes, emulsions, lipid ribbons or sheets, andmay be complexed with a variety of drugs and components which are knownto be pharmaceutically acceptable. In a preferred embodiment, the lipidconstruct is a liposome. The preferred liposome is unilamellar and has arelative size less than 200 nm. Common additional components in lipidconstructs include cholesterol and alpha-tocopherol, among others. Thelipid constructs may be used alone or in any combination which oneskilled in the art would appreciate to provide the characteristicsdesired for a particular application. In addition, the technical aspectsof lipid constructs and liposome formation are well known in the art andany of the methods commonly practiced in the field may be used for thepresent invention.

[0057] The methods described herein and the Nucleic Acid Ligandsidentified by such methods are useful for both therapeutic anddiagnostic purposes. Therapeutic uses include the treatment orprevention of diseases or medical conditions in human patients,specifically diseases or conditions caused by activation of theComplement System. The Complement System does not have to be the onlycause of the disease state, but it may be one of several factors, eachof which contributes to pathogenesis. Such diseases or conditionsinclude, but are not limited to, renal diseases, such as lupus nephritisand membranoproliferative glomerulonephritis (MPGN), membranousnephritis, IgA nephropathy; rheumatological diseases, such as rheumatoidarthritis, systemic lupus erythematosus (SLE), Behcet's syndrome,juvenile rheumatoid arthritis, Sjögren's syndrome and systemicsclerosis; neurological diseases, such as myasthenia gravis, multiplesclerosis, cerebral lupus, Guillain-Barré syndrome and Alzheimer'sdisease; dermatological diseases, such as Pemphigus/pemphigoid,phototoxic reactions, vasculitis and thermal bums; hematologicaldiseases, such as paroxysmal nocturnal hemoglobinuria (PNH), hereditaryerythroblastic multinuclearity with positive acidified serum lysis test(HEMPAS) and idiopathic thrombocytopenic purpura (ITP);biocompatibility/shock diseases, such as post-bypass syndrome, adultrespiratory distress syndrome (ARDS), catheter reactions, anaphylaxis,transplant rejection, pre-eclampsia, hemodialysis and platelet storage;vascular/pulmonary diseases, such as atherosclerosis, myocardialinfarction, stroke and reperfusion injury; allergies, such asanaphylaxis, asthma and skin reactions; infection, such as septic shock,viral infection and bacterial infection; and other conditions, such asatheroma, bowel inflammation, thyroiditis, infertility, paroxysmalnocturnal hemoglobinuria (PNH) and hemolytic anemia.

[0058] The Complement System can be inhibited at several points in theactivation cascade by targeting different components. Inhibition of C1qwould block the initiation by either antibody or non-antibodymechanisms. Antibodies activate C1q in many diseases including SLE,myasthenia gravis and arthritis. Non-antibody Complement Systemactivation occurs in many diseases including Alzheimer's disease,myocardial infarction and septic shock. Blocking C1q could prevent thecomplement-mediated tissue injury in these diseases.

[0059] The Complement can also be activated in the absence of antibodiesdirectly at the C3 stage. Activating surfaces including bacteria, virusparticles or damaged cells can trigger Complement System activation thatdoes not require C1q. An inhibitor of C3 could prevent Complement Systemactivation and damage in these situations.

[0060] In other instances the inhibition of C5 is most useful.Activation of the Complement System by either C1q or C3 mechanisms bothlead to activation of C5, so that inhibition of C5 could preventComplement System-mediated damage by either pathway. However, both C1qand C3 are important in normal defense against microorganisms and inclearance of immune components and damaged tissue, while C5 is mostlydispensable for this function. Therefore, C5 can be inhibited either fora short term or a long term and the protective role of Complement Systemwould not be compromised, whereas long term inhibition of C1q or C3 isnot desirable. Finally, the C5 fragments C5a and C5b directly cause themajority of tissue injury and disease associated with unwantedComplement System activation. Therefore, inhibition of C5 is the mostdirect way of producing therapeutic benefit.

[0061] In other instances, the activation of the Complement System isdesirable in the treatment or prevention of diseases or medicalconditions in human patients. For example, the activation of theComplement System is desirable in treating bacterial or viral infectionsand malignancies. In addition, the activation of the Complement Systemon T-cells prior to transplantation could prevent rejection of an organor tissue by eliminating the T-cells that mediate the rejection.

[0062] Furthermore, Nucleic Acid Ligands that bind to cell surfaceTargets could be made more efficient by giving them the ability toactivate the Complement System. Nucleic Acid binding would then bothinhibit a Target function and also eliminate the cell, for example, bymembrane attack complex lysis and cell clearance through opsonization.Nucleic Acid Ligands could activate the Complement System through eitherthe classical or the alternative pathways. C1q Nucleic Acid Ligands canbe conjugated to other structures that target a cell surface component.For example, C1q Nucleic Acid Ligands can be conjugated to antibodies tocell targets, cytokines, growth factors or a ligand to a cell receptor.This would allow the C1q Nucleic Acid Ligands to multimerize on thetargeted cell surface and activate the Complement System, therebykilling the cell.

[0063] The prototype classical pathway activators are immune aggregates,which activate the Complement System through binding to globular headgroups on the C1q component. Generally, binding of two or more Fcdomains to C1q is required; pentameric IgM is an especially efficientactivator. In contrast, Nucleic Acid Ligands can activate throughbinding at a separate site on the C1q collagen-like tail region. Thissite also binds to a variety of other non-antibody activators includingC-reactive protein, serum amyloid protein, endotoxin, β-amyloid peptide1-40 and mitochondrial membranes. As with immunoglobulin, thesenon-antibody activators need to be multimerized to activate.

[0064] Nucleic Acid Ligands that bind to sites on the collagen-likeregion of C1q may also become activators when aggregated. Such aComplement System-activating aggregate may be lytic if formed on a cellsurface, such as binding to a tumor-specific antigen (TSA) or to aleukocyte antigen. The extent of Nucleic Acid Ligand-mediated activationincreases with the extent of Nucleic Acid Ligand aggregation (i.e.,multiplicity of Nucleic Acid Ligand-C1q interaction). The ComplementSystem-mediated killing is especially specific if the Nucleic AcidLigands circulate as monomers which do not activate, but becomeactivators when they are multimerized on the targeted cell surface.

[0065] As with any Complement System activation, the extent andspecificity is determined by the amount of C3 deposited onto thetargeted cell. Deposited C3 forms an enzyme convertase that cleaves C5and initiates membrane attack complex formation. C3 is also theclassical serum opsonin for targeting phagocytic ingestion. Theprototype alternative pathway activators are repeating carbohydrateunits including bacterial and yeast cell walls, fucoidin and Sepharose,or glycolipids such as endotoxin or the glycocalyx. Nucleic Acid Ligandscould activate the alternative pathway by aggregating the C3 componenton the cell surface. Depositing C3 on a cell promotes Factor B bindingand alternative pathway C3 convertase formation. Binding of a NucleicAcid Ligand to C3 blocks binding of the inhibitor Factor H and preventsC3b decay. This would also increase C3 convertase formation andalternative path activation. Nucleic Acid Ligands to C3 may have thisactivity since heparin binds activated C3 and can promote alternativepathway activation. Binding of Nucleic Acid Ligands to C3 blocks bindingto C3 of the membrane-associated inhibitors CR1, CR2, MCP and DAF,preventing C3b convertase decay and stimulating alternative pathwayactivation. This alternative pathway mechanism can be as efficient asC1q-dependent activation in cell killing and lysis.

[0066] Nucleic Acid Ligand-mediated Complement System cell killing couldbe employed in several ways, for example, by: a) direct killing of tumorcells; b) lysis of targeted microorganisms or infected cells; and c)elimination of lymphocytes or lymphocyte subsets. Nucleic Acid Ligandscould replace antibodies currently used for these purposes.

[0067] Diagnostic utilization may include either in vivo or in vitrodiagnostic applications. The SELEX method generally, and the specificadaptations of the SELEX method taught and claimed herein specifically,are particularly suited for diagnostic applications. The SELEX methodidentifies Nucleic Acid Ligands that are able to bind targets with highaffinity and with surprising specificity. These characteristics are, ofcourse, the desired properties one skilled in the art would seek in adiagnostic ligand.

[0068] The Nucleic Acid Ligands of the present invention may beroutinely adapted for diagnostic purposes according to any number oftechniques employed by those skilled in the art. Diagnostic agents needonly be able to allow the user to identify the presence of a giventarget at a particular locale or concentration. Simply the ability toform binding pairs with the target may be sufficient to trigger apositive signal for diagnostic purposes. Those skilled in the art wouldalso be able to adapt any Nucleic Acid Ligand by procedures known in theart to incorporate a labeling tag in order to track the presence of suchligand. Such a tag could be used in a number of diagnostic procedures.The Nucleic Acid Ligands to C1q, C3 and C5 described herein mayspecifically be used for identification of the C1q, C3 or C5 protein.

[0069] The SELEX process provides high affinity ligands of a targetmolecule. This represents a singular achievement that is unprecedentedin the field of Nucleic Acids research. The present invention appliesthe SELEX procedure to the specific target C1q, which is part of thefirst component (C1) of the classical pathway of Complement Systemactivation, to the specific target C3, which is part of both theclassical and alternative pathway, and to the specific target C5, whichis part of the terminal pathway. In the Example section below, theexperimental parameters used to isolate and identify the Nucleic AcidLigands to C1q, C3 and C5 are described.

[0070] In order to produce Nucleic Acids desirable for use as apharmaceutical, it is preferred that the Nucleic Acid Ligand (1) bindsto the target in a manner capable of achieving the desired effect on thetarget; (2) be as small as possible to obtain the desired effect; (3) beas stable as possible; and (4) be a specific ligand to the chosentarget. In most situations, it is preferred that the Nucleic Acid Ligandhave the highest possible affinity to the Target.

[0071] Pharmaceutical agents, which include, but are not limited to,small molecules, antisense oligonucleotides, nucleosides, andpolypeptides can activate the Complement System in an undesirablemanner. Nucleic Acid Ligands to Complement System Proteins could be usedas a prophylactic by transiently inhibiting the Complement System, sothat a pharmaceutical agent could be administered and achieve atherapeutically effective amount without eliciting the undesirable sideeffect of activating the Complement System.

[0072] In co-pending and commonly assigned U.S. patent application Ser.No. 07/964,624, filed October 21, 1992, now U.S. Pat. No. 5,496,938,(the '938 Patent), methods are described for obtaining improved NucleicAcid Ligands after SELEX has been performed. The '938 Patent, entitled“Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” is specificallyincorporated herein by reference in its entirety.

[0073] In the present invention, SELEX experiments were performed inorder to identify RNA with specific high affinity for C1q, C3 and C5from a degenerate library containing 30 or 50 random positions (30N or50N). This invention includes the specific RNA ligands to C1q shown inTable 2 (SEQ ID NOS:5-20) and Table 6 (SEQ ID NOS:84-155), identified bythe method described in Examples 2 and 6, the specific RNA ligands to C3shown in Table 3 (SEQ ID NOS:21-46), identified by method described inExample 3, and the specific RNA ligands to C5 shown in Table 4 (SEQ IDNOS:47-74), Table 5 (SEQ ID NOS:76-83), Table 8 (SEQ ID NOS:75,160-162), Table 10 (SEQ ID NOS:163-189), Table 12 (SEQ ID NOS:190-192),Table 13 (SEQ ID NOS:194-196) and FIGS. 5A-B (SEQ ID NOS:160 and 193)identified by methods described in Examples 4, 9, 10 and 11. Thisinvention further includes RNA ligands to C1q, C3 and C5 which inhibitthe function of C1q, C3 and C5. The scope of the ligands covered by thisinvention extends to all Nucleic Acid Ligands of C1q, C3 and C5,modified and unmodified, identified according to the SELEX procedure.More specifically, this invention includes Nucleic Acid sequences thatare substantially homologous to the ligands shown in Tables 2-6, 8, 10and 12-13 and FIGS. 5A-B (SEQ ID NOS:5-155 and 160-196). Bysubstantially homologous, it is meant a degree of primary sequencehomology in excess of 70%, most preferably in excess of 80%, and evenmore preferably in excess of 90%, 95% or 99%. The percentage of homologyas described herein is calculated as the percentage of nucleotides foundin the smaller of the two sequences which align with identicalnucleotide residues in the sequence being compared when 1 gap in alength of 10 nucleotides may be introduced to assist in that alignment.A review of the sequence homologies of the ligands of C1q shown in Table2 (SEQ ID NOS:5-20) and Table 6 (SEQ ID NOS:84-155) shows that sequenceswith little or no primary homology may have substantially the sameability to bind C1q. Similarly, a review of the sequence homologies ofthe ligands of C3 shown in Table 3 (SEQ ID NOS:21-46) shows thatsequences with little or no primary homology may have substantially thesame ability to bind C3. Similarly, a review of the sequence homologiesof the ligands of C5 shown in Table 4 (SEQ ID NOS:47-74), Table 5 (SEQID NOS:76-83), Table 8 (SEQ ID NOS:75, 160-162), Table 10 (SEQ IDNOS:163-189), Table 12 (SEQ ID NOS:190-192), Table 13 (SEQ IDNOS:194-196) and FIGS. 5A-B (SEQ ID NOS:160 and 193) shows thatsequences with little or no primary homology may have substantially thesame ability to bind C5. For these reasons, this invention also includesNucleic Acid Ligands that have substantially the same structure andability to bind C1q as the Nucleic Acid Ligands shown in Table 2 (SEQ IDNOS:5-20) and Table 6 (SEQ ID NOS:84-155), Nucleic Acid Ligands thathave substantially the same structure and ability to bind C3 as theNucleic Acid Ligands shown in Table 3 (SEQ ID NOS:21-46) and NucleicAcid Ligands that have substantially the same structure and ability tobind C5 as the Nucleic Acid Ligands shown in Table 4 (SEQ ID NOS:47-74),Table 5 (SEQ ID NOS:76-83), Table 8 (SEQ ID NOS:75, 160-162), Table 10(SEQ ID NOS:163-189), Table 12 (SEQ ID NOS:190-192), Table 13 (SEQ IDNOS:194-196) and FIGS. 5A-B (SEQ ID NOS:160 and 193). Substantially thesame ability to bind C1q, C3 or C5 means that the affinity is within oneor two orders of magnitude of the affinity of the ligands describedherein. It is well within the skill of those of ordinary skill in theart to determine whether a given sequence—substantially homologous tothose specifically described herein—has substantially the same abilityto bind C1q, C3 or C5.

[0074] The invention also includes Nucleic Acid Ligands that havesubstantially the same postulated structure or structural motifs.Substantially the same structure or structural motifs can be postulatedby sequence alignment using the Zukerfold program (see Zucker (1989)Science 244:48-52). As would be known in the art, other computerprograms can be used for predicting secondary structure and structuralmotifs. Substantially the same structure or structural motif of NucleicAcid Ligands in solution or as a bound structure can also be postulatedusing NMR or other techniques as would be known in the art.

[0075] One potential problem encountered in the therapeutic,prophylactic and in vivo diagnostic use of Nucleic Acids is thatoligonucleotides in their phosphodiester form may be quickly degraded inbody fluids by intracellular and extracellular enzymes such asendonucleases and exonucleases before the desired effect is manifest.Certain chemical modifications of the Nucleic Acid Ligand can be made toincrease the in vivo stability of the Nucleic Acid Ligand or to enhanceor to mediate the delivery of the Nucleic Acid Ligand. See, e.g., U.S.patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”now abandoned (see also U.S. Pat. No. 5,660,985) and U.S. patentapplication Ser. No. 08/434,465, filed May 4, 1995, entitled “NucleicAcid Ligand Complexes,” which are specifically incorporated herein byreference in their entirety. Modifications of the Nucleic Acid Ligandscontemplated in this invention include, but are not limited to, thosewhich provide other chemical groups that incorporate additional charge,polarizability, hydrophobicity, hydrogen bonding, electrostaticinteraction, and fluxionality to the Nucleic Acid Ligand bases or to theNucleic Acid Ligand as a whole. Such modifications include, but are notlimited to, 2′-position sugar modifications, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atexocyclic amines, substitution of 4-thiouridine, substitution of 5-bromoor 5-iodo-uracil, backbone modifications, phosphorothioate or alkylphosphate modifications, methylations, unusual base-pairing combinationssuch as the isobases isocytidine and isoguanidine and the like.Modifications can also include 3′ and 5′ modifications such as capping.

[0076] Where the Nucleic Acid Ligands are derived by the SELEX method,the modifications can be pre- or post-SELEX modifications. Pre-SELEXmodifications yield Nucleic Acid Ligands with both specificity for theirSELEX Target and improved in vivo stability. Post-SELEX modificationsmade to 2′-OH Nucleic Acid Ligands can result in improved in vivostability without adversely affecting the binding capacity of theNucleic Acid Ligand. The preferred modifications of the Nucleic AcidLigands of the subject invention are 5′ and 3′ phosphorothioate cappingand/or 3′-3′ inverted phosphodiester linkage at the 3′ end. In onepreferred embodiment, the preferred modification of the Nucleic AcidLigand is a 3′-3′ inverted phosphodiester linkage at the 3′ end.Additional 2′-fluoro (2′-F) and/or 2′-amino (2′-NH₂) and/or 2′-O-methyl(2′-OMe) modification of some or all of the nucleotides is preferred.Described herein are Nucleic Acid Ligands that were 2′-NH₂ modified or2′-F modified and incorporated into the SELEX process. Further describedherein are 2′-F modified Nucleic Acid Ligands derived from the SELEXprocess which were modified to comprise 2′-OMe purines in post-SELEXmodifications.

[0077] Other modifications are known to one of ordinary skill in theart. Such modifications may be made post-SELEX (modification ofpreviously identified unmodified ligands) or by incorporation into theSELEX process.

[0078] As described above, because of their ability to selectively bindC1q, C3 and C5, the Nucleic Acid Ligands to C1q, C3 and C5 describedherein are useful as pharmaceuticals. This invention, therefore, alsoincludes a method for treating Complement System-mediated diseases byadministration of a Nucleic Acid Ligand capable of binding to aComplement System Protein or homologous proteins. Certain diseases orconditions such as Alzheimer's disease or myocardial infarction activateC1q through the collagen-like region. In Alzheimer's disease, β-amyloidactivates C1q. Structures in heart muscle that are exposed duringmyocardial infarction such as intermediate filaments, mitochondrialmembranes or actin activate C1q. Nucleic Acid Ligands to C3 or to C5could also inhibit Complement System activation in Alzheimer's diseaseor myocardial infarction, whether the Complement System is activatedthrough C1q by antibody or non-antibody mechanisms, or independent ofC1q through the alternative pathway. Thus, the Nucleic Acid Ligands ofthe present invention may be useful in treating Alzheimer's disease ormyocardial infarction.

[0079] Therapeutic compositions of the Nucleic Acid Ligands may beadministered parenterally by injection, although other effectiveadministration forms, such as intraarticular injection, inhalant mists,orally active formulations, transdermal iontophoresis or suppositoriesare also envisioned. One preferred carrier is physiological salinesolution, but it is contemplated that other pharmaceutically acceptablecarriers may also be used. In one preferred embodiment, it is envisionedthat the carrier and the Nucleic Acid Ligand constitute aphysiologically-compatible, slow release formulation. The primarysolvent in such a carrier may be either aqueous or non-aqueous innature. In addition, the carrier may contain otherpharmacologically-acceptable excipients for modifying or maintaining thepH, osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution, or odor of the formulation. Similarly, the carrier maycontain still other pharmacologically-acceptable excipients formodifying or maintaining the stability, rate of dissolution, release orabsorption of the ligand. Such excipients are those substances usuallyand customarily employed to formulate dosages for parentaladministration in either unit dose or multi-dose form.

[0080] Once the therapeutic composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or dehydrated or lyophilized powder. Such formulations may be storedeither in a ready to use form or requiring reconstitution immediatelyprior to administration. The manner of administering formulationscontaining Nucleic Acid Ligands for systemic delivery may be viasubcutaneous, intramuscular, intravenous, intranasal or vaginal orrectal suppository.

[0081] The following Examples are provided to explain and illustrate thepresent invention and are not intended to be limiting of the invention.These Examples describe the use of SELEX methodology to identify highaffinity RNA ligands to C1q, C3 and C5. Example 1 describes the variousmaterials and experimental procedures used in Examples 2, 3, 4 and 6.Example 2 describes the generation of 2′-NH₂ RNA ligands to C1q. Example3 describes the generation of 2′-F Nucleic Acid Ligands of ComplementSystem Protein C3. Example 4 describes the generation of 2′-F NucleicAcid Ligands of Complement System Protein C5. Example 5 describes theactivation of the Complement System through C1q ligands. Example 6describes the generation of 2′-F RNA ligands to C1q. Example 7 describesan assay for hemolytic inhibition for 2′-F RNA ligands to C5. Example 8describes an assay for inhibition of C5a release by a Nucleic AcidLigand (clone C6) to Human C5. Example 9 describes boundary experimentsperformed to determine the minimum binding sequence for Nucleic AcidLigands to Human C5. Example 10 describes a Biased SELEX experimentperformed to improve Nucleic Acid Ligand affinity, using a 42 mertruncated sequence of clone C6 as the random sequence in the template.Example 11 describes the results of 2′-OMe purine substitutions in aHuman C5 Nucleic Acid Ligand in an interference assay. Example 12describes the structure of a 38 mer truncate of a Nucleic Acid Ligand tohuman C5. Example 13 describes a hemolytic assay of 2′-OMe purinesubstituted Nucleic Acid Ligands to human C5.

Example 1

[0082] Experimental Procedures

[0083] This example provides general procedures followed andincorporated in Examples 2, 3, 4 and 6 for the identification of 2′-NH₂and 2′-F RNA ligands to C1q, and 2′-F ligands to C3 and C5.

[0084] A. Biochemicals

[0085] C1q, C3, C5 and C4-deficient guinea pig sera were obtained fromQuidel (San Diego, Calif.). Bovine serum albumin (BSA), rabbit anti-BSA,CRP, SAP and β-amyloid peptides 1-40 and 1-42 were obtained from Sigma(St. Louis, Mo.). Nucleotides GTP, ATP and deoxynucleotides wereobtained from Pharmacia (Uppsala, Sweden). Taq polymerase was obtainedfrom Perkin-Elmer (Norwalk, Conn.). Modified nucleotides 2′-NH₂-CTP and2′-NH₂-UTP, and 2′-F-CTP and 2′-F-UTP, were prepared as described inJellinek et al. (1995) Biochem. 34:11363. Avian reverse transcriptasewas obtained from Life Sciences (St. Petersburg, Fla.) and T7 RNApolymerase from USB (Cleveland, Ohio.). Nitrocellulose filters wereobtained from Millipore (Bedford, Mass.). All chemicals were the highestgrade available.

[0086] B. RNA SELEX Procedures

[0087] The SELEX procedure has been described in detail in the SELEXPatent Applications (see also Jellinek et al. (1995) Biochem. 34:11363;Jellinek et al. (1994) Biochem. 33:10450). Briefly, a DNA template wassynthesized with a 5′ fixed region containing the T7 promoter, followedby a 30N or a 50N stretch of random sequence, and then with a 3′-fixedregion (Table 1; SEQ ID NOS:1 and 156). For the initial round of theSELEX process, 1 nmole (˜10¹⁴ unique sequences) of RNA (Table 1; SEQ IDNOS:2 and 157) was in vitro transcribed by T7 polymerase (Milligan etal. (1987) Nucleic Acids Res. 12:785) using mixed GTP/ATP and2′-NH₂-CTP/UTP or 2′-F-CTP/UTP nucleotides, and with the addition ofα-[³²P]-ATP. For this and subsequent rounds of the SELEX process, theRNA was purified by electrophoresis on 8% acrylamide gels with 7 M urea,10 mM Tris-Borate, 2 mM EDTA, pH 8.3 running buffer. Afterautoradiography, the band containing labeled, modified RNA transcriptwas excised and frozen at −70° C., then 400 μL of 100 mM NaCl, 2 mM EDTAwas added, the gel was mashed, and the slurry was spun through 2 cm ofglass-wool (Rnase-free—Alltech Associates, Deerfield, Ill.) and twonitrocellulose filters. The RNA was precipitated by addition of ⅕ vol of6.6 M NH₄OAc, pH 7.7, plus 2 vol of ethanol. The pellet was washed twicewith 80% ethanol, and taken to dryness. The dry RNA pellet was dissolvedin phosphate buffered saline (Sambrook et al. (1989) Molecular Cloning.A laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,NY) containing 1 mM MgCl₂ (MgPBS).

[0088] For each round of the SELEX process, the RNA was incubated withC1q, C3 or C5 in MgPBS for 10 minutes at 37° C. Then the sample wasfiltered through a 43 mm nitrocellulose filter, and the filter waswashed with 10 mL of MgPBS. For some rounds, the diluted RNA waspre-soaked with nitrocellulose filters overnight to reduce background.Four samples were run in parallel for most rounds with lesser amounts(chosen to be in suitable range to measure binding) of both RNA and C1q,C3 or C5 to measure binding Kd for each sample. In addition, at eachround, a sample of RNA was filtered without protein to determinebackground.

[0089] Filters were air-dried, sliced into strips, counted, and thenextracted for 60 minutes at 37° C. with 400 μL of 1% SDS, 0.5 mg/mLProteinase K (Boehringer Mannheim, Indianapolis, Ind.), 1.5 mM DTT, 10mM EDTA, 0.1 M Tris, pH 7.5, with addition of 40 μg tRNA carrier. Theaqueous RNA was extracted with phenol, phenol/chloroform (1:1), andchloroform and then precipitated following addition of NH₄OAc/EtOH asabove. The RNA was reverse transcribed in a volume of 50 μL for between1 hour and overnight. The DNA was PCR amplified with specific primers(Table 1; SEQ ID NOS:3-4) in a volume of 500 μL for 12-14 cycles, andthen phenol/chloroform extracted and NaOAc/EtOH precipitated. The DNApellet was taken up in H₂O, and an aliquot was T7 transcribed for thenext round of the SELEX process.

[0090] C. Cloning DNA from the 12^(th) or the 14^(th) round was PCRamplified with primers which also contained a ligation site tofacilitate cloning. The DNA was cloned into a pUC9 vector, and colonieswere picked for overnight growth and plasmid mini-preps (PERFECTprep,5′-3′, Boulder, Colo.). The purified plasmids were PCR amplified withoriginal 3′ and 5′ primers (as above), and products were analyzed byagarose gel electrophoresis (Sambrook et al. (1989) Molecular Cloning. Alaboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). DNA was T7 transcribed with α-[³²P]-ATP to prepare radiolabeledRNA for binding analysis and without radiolabel to prepare RNA forinhibition studies.

[0091] D. Sequencing Plasmids purified using the PERFECTprep kit weresequenced with ABI dRhodamine Terminator cycling kit (Perkin-Elmer).Samples were sequenced on the ABI Prism 377 DNA Sequencer.

[0092] E. Binding Assays

[0093] Individual cloned DNA was T7 transcribed with α-[³²P]-ATP and thefull length [³²P]-2′-NH₂-RNA or 2′-F-RNA was gel-purified (as above).RNA was suspended at approximately 5,000 cpm per 30 μL sample (<10 pM),and aliquots were incubated with various concentrations of C1q, C3 or C5in MgPBS for 10 minutes at 37° C. Samples were then filtered throughnitrocellulose, the filters washed with buffer and dried under aninfrared lamp, and counted with addition of scintillation fluid(Ecoscint A, National Diagnostics, Atlanta, Ga.). A background sample ofRNA alone was run in parallel. To measure inhibition of ligand bindingto C1q, the RNA Nucleic Acid Ligand plus C1q plus inhibitor (e.g., theA-chain residue 14-26 site, SAP, β-amyloid peptide, CRP) were incubatedfor 10 minutes at 37° C., and then filtered. Filters were washed andcounted.

[0094] RNA ligand binding to C1q was also measured in the presence ofimmune-complexes, which would block the binding of ligands to C1qhead-groups. Immune complexes (IC) were formed by mixing 620 μg BSA atequivalence with 1 mL of rabbit anti-BSA (Sigma, St. Louis, Mo.) plusPEG 8000 added to 1% final concentration, and then the samples wereincubated overnight at 4° C. The IC were pelleted by microfugation at12,000 rpm for 10 minutes, washed five times with PBS, and suspended in1 mL of MgPBS. For measurement of C1q RNA clone binding to C1q-immunecomplexes (C1q-IC), 20 μL of the purified [³²P]-RNA plus 20 μL of the ICwere mixed with 20 μL of C1q at various concentrations at between 10⁻¹¹and 10⁻⁷ M in MgPBS plus 1% Triton. Samples were incubated for 30minutes at room temperature, microfuged, and the pellets andsupernatants counted.

[0095] F. Hemolytic Assays

[0096] Complement System consumption was measured by C4 hemolytic assayas described (Gaither et al. (1974) J. Immunol. 113:574). All sampleswere diluted and the assay run in veronal-buffered saline containingcalcium, magnesium and 1% gelatin (GVB⁺⁺-complement buffer). Formeasurement of C4 consumption by β-amyloid peptide consumption, thepeptide was added at 250 μg/mL to a 1/8 dilution of whole human serumand then incubated for 60 minutes at 37° C. The sample was then dilutedfor assay of C4 hemolytic activity. For assay of inhibition of β-amyloidpeptide mediated complement consumption by C1 q 2′-NH₂-RNA clones, theC1q RNA Nucleic Acid Ligand was included in the initial β-amyloidpeptide-whole human serum incubation mixture, and then C4 amountsassayed as above.

[0097] Complement System inhibition by C5 Nucleic Acid Ligands wasmeasured using human serum and antibody-coated sheep red blood cells.The red blood cells were incubated with a 1:40 dilution of fresh humanserum and with serial dilutions of C5 ligand for 30 minutes at 37° C.Dilutions of serum and ligand were made in complement buffer (seeprevious paragraph). After incubation the samples were then diluted with4° C. buffer containing EDTA to stop the reaction, and the hemoglobinrelease was quantitated from the optical density at 412 nm.

Example 2

[0098] 2′-NH₂ RNA Ligands to C1q

[0099] A. RNA SELEX

[0100] The pool of random 50N7-2′-NH₂ RNA bound to C1q by nitrocellulosefilter assay with a K_(d) of 2.3 μM. For round 1 of the SELEX process,the C1q concentration was between 0.156-1.25 μM and the RNAconcentration was 15 μM. Throughout the SELEX process, the RNAconcentrations were maintained at approximately 10-fold greater than theconcentration of C1q, which was reduced at each round with a final round14 C1q concentration of 136 pM. Background binding of RNA tonitrocellulose filters remained low throughout the SELEX procedure, inpart because RNA was pre-adsorbed with nitrocellulose filters. Thebinding of pool RNA to C1q improved at each round. The evolved round 14pool 2′-NH₂ RNA bound C1q with a K_(d)=670 pM, yielding an overallimprovement in binding K_(d) of 3400-fold.

[0101] Bulk RNA was then cloned for sequence determination andevaluation of binding. Through comparison of binding at 0.1 and 0.5 nMC1q, individual clones were ranked, and clones with C1q binding abovebackground were sequenced and are shown in Table 2 (SEQ ID NOS:5-20).Family 1 contained 12 of the 19 total sequences. Family 2 containedthree sequences. Both Family 3 and Family 4 contained two sequences.Both Family 1 and Family 2 sequences contain G-rich regions and bothhave the repeated sequence motifs GGAG and GGUG. The identity andhomology of Family 1 members is greatest in the 5′ half, which isG-rich. The C-rich 3′ half has only short stretches of sequencehomology, and these are shown only with inclusion of large gap regions.Sequences from all families can be folded to give stem-loop structureswith extensive Watson-Crick base-pairing. Full binding curves for thehighest affinity ligands yielded a K_(d) range from 290 pM to 3.9 nM;the high affinity ligands were found in all four sequence families. Allof the binding curves were monophasic. The binding maximum is not 100%because of variable amounts of nucleic acid alterations taking placeduring purification. This is known because usually ligands can be boundto protein, extracted, and then re-bound, and give maximum bindingapproaching 100% (data not shown).

[0102] B. Competition

[0103] 2′-NH₂ RNA ligands from different families interact with the sameor overlapping sites on C1q, as shown by cross-competition. This site ison the collagen-like region, at or near the A-chain 14-26 residue site(Jiang et al. (1994) J. Immunol. 152:5050) as shown by two lines ofevidence. First, C1q when bound to IC still binds the ligand #50 (SEQ IDNO:12); binding to immunoglobulin Fc would block the head region, butleave the collagen-like tail available, suggesting that nucleic acidligands derived by the SELEX process are bound to the tail. Second, andmore direct, ligand #50 is competed by proteins which are known to bindthe A-chain residue 14-26 site, including SAP, β-amyloid peptide andCRP. Finally, ligand #50 is competed by a peptide that has the sameamino acid sequence as residues 14-26 on the A-chain. This result isfurther supported by results for hemolytic inhibition as describedbelow.

[0104] C. Consumption

[0105] Binding of a nucleic acid ligand derived by the SELEX process tothe A-chain 14-26 amino acid site could activate C1q or alternatively,SELEX-derived nucleic acid ligands could inhibit the binding of othermolecules and prevent C1q activation. This was tested by measuring C4consumption in serum after incubation with a 2′-NH₂ SELEX-derivednucleic acid ligands, or after incubation with a known C1q activatortogether with a 2′-NH₂ nucleic acid ligand. The SELEX-derived nucleicacid ligands when incubated in serum do not consume C4, and thus are notC1q activators. Nor do these ligands at this concentration inhibit serumlysis of antibody-coated sheep erythrocytes, which would occur ifligands bound near the C1q head groups (data not shown). The ligands doinhibit C4 consumption by another C1q activator, the β-amyloid 1-40peptide. This peptide is known to activate C1q through binding at theA-chain 14-26 residue site; therefore, this inhibition confirms thatSELEX-derived nucleic acid ligands bind at this A-chain site. Controlligands from the SELEX process that did not bind C1q by nitrocelluloseassay were also ineffective in blocking the β-amyloid 1-40 peptide C1qactivation.

Example 3

[0106] 2′-Fluoro Nucleic Acid Ligands of Complement System Protein C3

[0107] In order to generate ligands to complement protein C3, a libraryof about 10¹⁴ RNA was generated that contained 30 nucleotides ofcontiguous random sequence flanked by defined sequences. In thisexperiment, 30N random nucleotides of the initial Candidate Mixture werecomprised of 2′-F pyrimidine bases. The rounds of selection andamplification were carried out as described in Example 1 using art-knowntechniques. In round 1 the 30N7-2′-F-RNA and C3 were both incubated at 3μM. There was barely detectable binding at this round. Both the RNA andC3 concentrations were decreased during the SELEX procedure. Sequencesderived from the SELEX procedure are shown in Table 3 (SEQ IDNOS:21-46).

Example 4

[0108] 2′-Fluoro Nucleic Acid Ligands of Complement System Protein C5

[0109] In order to generate ligands to human complement protein C5, alibrary of about 10¹⁴ RNA was generated that contained 30 nucleotides ofcontiguous random sequence flanked by defined sequences. In thisexperiment, the 30N random nucleotides of the initial Candidate Mixturewere comprised of 2′-F pyrimidine bases. Briefly, a DNA template wassynthesized with a 5′-fixed region containing the T7 promoter, followedby a 30N stretch of random sequence, and then with a 3′-fixed region(Table 1; SEQ ID NO:1). The rounds of selection and amplification werecarried out as described in Example 1 using art-known techniques. Theinitial rounds of the SELEX experiment were set up with highconcentrations of 2′-F RNA (7.5 μM) and protein (3 μM), as the bindingof C5 to unselected RNA was quite low. The SELEX experiment was designedto promote binding of RNA at the C5a-C5b cleavage site. RNA and C5 wereincubated together with small amounts of trypsin, with the reasoningthat limited trypsin treatment of C5 produces a single site cleavage andgenerates C5a-like activity (Wetsel and Kolb (1983) J. Exp. Med.157:2029). This cleavage led to a slight increase in random RNA binding.Enhanced RNA binding associated structurally with exposure of theC5a-like domain could evolve Nucleic Acid Ligands that bind near the C5convertase site and could interfere with or inhibit C5 cleavage. TheSELEX experiment was performed simultaneously to both the native and tothe mildly-trypsinized protein, so that Nucleic Acid Ligand evolutionwould pick the highest affinity winner. With this procedure the highestaffinity winner against the multiple protein species would be evolved,and multiple aptamers and specific aptamers might be obtained out of asingle SELEX experiment.

[0110] For each round of the SELEX process, the procedure was performedin parallel in separate tubes with approximately 5-fold excess of RNAeither in buffer alone or with addition of trypsin at between 0.3 and0.0001 mg/mL. Samples were incubated in MgPBS for 45 minutes at 37° C.,and then filtered through nitrocellulose. The filters were washed, driedand counted, extracted, reverse-transcribed, then PCR amplified andfinally T7 transcribed in vitro into RNA using mixed GTP/ATP and2′-F-CTP/UTP nucleotides and α-[³²P]-ATP. RNA was purified byelectrophoresis in 8% acrylamide gels with 7M urea and Tris-Borate EDTAbuffer (TBE). RNA was isolated and precipitated with NH₄OAc/ethanol, andthen dissolved in phosphate-buffered saline containing 1 nM MgCl₂(MgPBS). Filters with the highest binding were carried forward. At theend of each round, all of the RNA that bound to the protein (either withor without trypsin) was pooled. The protein and RNA concentrations ateach round were reduced, with final concentrations of 2.5 nM and 10 nMrespectively. Trypsin was added at concentrations between 0.3 and 0.0001μg/mL. Background binding was monitored at each round, and starting atround four the transcribed RNA was presoaked overnight withnitrocellulose filters prior to the SELEX rounds to reduce background.

[0111] Based on binding of RNA to native C5 by nitrocellulose assay,round twelve DNA was cloned and sequences were obtained as shown inTable 4 (SEQ ID NOS:47-74). Sequences were grouped according to homologyand function. Group I sequences are highly homologous and might havearisen by PCR mutation from a single original sequence. Bindingaffinities of the Group I Nucleic Acid Ligands are very similar and areshown in Table 7. Group II Nucleic Acid Ligands generally bound withsimilar affinity to Group I Nucleic Acid Ligands, although some weakbinders were also present. Group II sequences and length are morediverse than Group I Nucleic Acid Ligands. The C5 Nucleic Acid Ligandsdo not bind other complement components including C1q, C3, or factors B,H, or D.

[0112] Nucleic Acid Ligands from each family were also assayed forinhibition of rat Complement System activity (Table 5; SEQ IDNOS:76-83). Nucleic Acid Ligands from Family I and Family III inhibitedrat complement, whereas a Nucleic Acid Ligand from Family II did not. Aninhibitory Nucleic Acid Ligand can be used to inhibit Complement Systemactivity in various rat disease models including, but not limited to,myasthenia gravis, myocardial infarction, glomerulonephritis, ARDS,arthritis and transplantation.

Example 5

[0113] Activation of the Complement System through C1q Nucleic AcidLigands

[0114] Oligonucleotides can activate both classical and alternativepathways. Particularly, poly-G oligonucleotides which can form G-quartetstructures and can interact with the C1q collagen-like region are ableto form high molecular weight aggregates, which both bind and activateC1q. Phosphorothioate oligonucleotides, which have increasednon-specific binding as compared with phosphodiester oligonucleotides,are also efficient Complement System activators, particularly poly-Gcontaining phosphorothioate oligonucleotides. Results foroligonucleotide activation of solution phase Complement are shown belowwhere classical pathway activation is measure by the release of C4dfragment by ELISA (Quidel, San Diego, Calif.), and alternative pathwayactivation is measure by Bb ELISA (Quidel, San Diego, Calif.). Althoughthese pathways are separate, there is evidence to suggest thatoligonucleotide activation of both pathways is C1q dependent. [C4d] μg[Bb] μg Sample (Class.) (Altern.) Poly-AG Random Co-Polymer 8.1 18.9Poly-G Random Co-Polymer 1.2 29.3 Poly-I Random Co-Polymer 0 14.7 Poly-ARandom Co-Polymer 0 0 Poly-U Random Co-Polymer 0 1.8 Poly-C RandomCo-Polymer 0 2.5 Phosphorothioate OligonucleotidesGGCGGGGCTACGTACCGGGGCTTTGTAAAACCCCGCC −7.1 32.4 SEQ ID NO: 197CTCTCGCACCCATCTCTCTCCTTCT 0.0 3.9 SEQ ID NO: 198 BSA-anti-BSA ImmuneComplexes 8.0 11.9 β-Amyloid Peptide 2.7 n/d Fucoidan SulfatedCarbohydrate 27 buffer 0.0 0.0

[0115] Complement System activation is also initiated on the erythrocytemembrane and is tested by hemolytic assays. Known activators, including2′-OH poly-G and phosphorothioate oligonucleotides, as well as potentialactivators such as multimerized C1q Nucleic Acid Ligands and small(e.g., 15-mer) 2′-F poly-G oligonucleotides are coated on sheeperythrocytes and subsequent lysis of the erythrocytes by serumcomplement is measured. Methods of coating oligonucleotides and NucleicAcid Ligands on cells include passive adsorption, chemical conjugation,streptavidin-biotin coupling and specific Nucleic Acid binding.Following treatment with fresh rat or human serum, the deposition ofcomplement components on the cell, membrane damage and lysis aremeasured by standard methods as would be known by one of skill in theart.

[0116] A. Aggregation of C1q Nucleic Acid Ligands

[0117] C1q Nucleic Acid Ligands are dimerized using chemicalcross-linkers of various lengths. Alternatively, Nucleic Acid Ligandmonomers are biotinylated and then multimerized with streptavidin. Eachof these multimers are tested for complement activation and lysis oferythrocytes.

[0118] The addition of poly-G sequence to C1q Nucleic Acid Ligandsprovides additional binding ability and increases the ability of theoligonucleotide to activate the Complement System. In addition, shortpoly-G sequences on individual C1 q Nucleic Acid Ligands can interact toform higher order structures, which serve to multimerize the C1q NucleicAcid Ligands and cause activation.

[0119] B. Lysis of Erythrocytes and Leukocytes

[0120] Nucleic Acid Ligands that promote erythrocyte lysis are tested onnucleated cells, including lymphocytes and tumor cells. Nucleated cellshave mechanisms of complement resistance that erythrocytes lack. Forexample, nucleated cells can shed antigens, bleb off membrane vesiclescontaining the complement components and express increased levels ofcomplement inhibitors as compared with erythrocytes and may up-regulateprotective mechanisms upon initial complement attack. As high levels ofactivation are important for cell killing, activators are compared foramount of Complement System component deposition and extent of membranedamage. Also, different types and sources of tumor cells and lymphocytesare tested to determine if susceptibility is cell-type specific.

[0121] Nucleic Acid Ligands can be generated for virtually any target asdescribed in the SELEX Patent Applications. Nucleic Acid Ligands toL-Selectin have been generated (See U.S. patent application Ser. No.08/479,724, filed Jun. 7, 1995, entitled “High Affinity Nucleic AcidLigands to Lectins,” now U.S. Pat. No. 5,780,228, which is incorporatedherein by reference in its entirety). The diversity of lectin mediatedfunctions provides a vast array of potential therapeutic targets forlectin antagonists. For example, antagonists to the mammalian selecting,a family of endogenous carbohydrate binding lectins, may havetherapeutic applications in a variety of leukocyte-mediated diseasestates. Inhibition of selectin binding to its receptor blocks cellularadhesion and consequently may be useful in treating inflammation,coagulation, transplant rejection, tumor metastasis, rheumatoidarthritis, reperfusion injury, stroke, myocardial infarction, bums,psoriasis, multiple sclerosis, bacterial sepsis, hypovolaemic andtraumatic shock, acute lung injury and ARDS. The coupling of C1q NucleicAcid Ligands to L-Selectin Nucleic Acid Ligands makes the L-SelectinNucleic Acid Ligand more efficient by promoting cell killing at thetarget. C1q Nucleic Acid Ligands are coupled to L-Selectin Nucleic AcidLigands, and the conjugates are tested for leukocyte lysis as describedabove. Also, Nucleic Acid Ligands to other cell surface targets,antibodies to all targets that do not themselves activate complement,cytokines, growth factors, or a ligand to a cell receptor could becoupled to a C1q Nucleic Acid Ligand and used for cell killing.

[0122] C. In Vivo Testing of Complement Activation

[0123] Nucleic Acid Ligand-mediated Complement System activation istested in animals to evaluate in vivo Nucleic Acid Ligand action.Erythrocytes and/or lymphocytes are coated with Nucleic Acid Ligands andinjected into rats to test cell killing and lysis in vivo. ActivatingNucleic Acid Ligands are also coupled to a Mab that does not activatethe Complement System, where the antibody is directed against a rat cellantigen (e.g., lymphocyte antigen). These cells are then coated with theNucleic Acid Ligand-antibody conjugate and injected into rats.Alternatively, the Nucleic Acid Ligand-antibody conjugate is injecteddirectly into the rat and then in vivo leukocyte killing is measured.

[0124] It is also possible that C1q Nucleic Acid Ligands cross-reactwith non-human C1q, and non-human C1q could be used for in vivo assays.C1q Nucleic Acid Ligands are tested against species such as mouse, ratand rabbit C1q. C1q is purified from serum and cross-reactivity with C1qNucleic Acid Ligands is tested by nitrocellulose binding assay.Alternatively, C1q is bound to immune complexes which are added to serumand then C1q Nucleic Acid Ligand binding to the aggregate is tested. IfNucleic Acid Ligands are species-specific, then rat serum is depleted ofrat C1q by continuous perfusion over a Ig-Sepharose column, and theserum is reconstituted with human C1 q by methods known to one of skillin the art. These reconstituted animals are then used to test C1qNucleic Acid Ligands for targeted Complement System activation and cellkilling.

Example 6

[0125] 2′-Fluoro RNA Ligands of Complement System Protein C1q

[0126] A. RNA SELEX

[0127] The pool of random 30N7-2′-F RNA bound to C1q by nitrocellulosefilter assay with a K_(d) of 2.3 μM. For round 1 of the SELEX process,the C1q concentration was between 0.156-1.25 μM and the RNAconcentration was 15 μM. Throughout the SELEX process, the RNAconcentrations were maintained at approximately 10-fold greater than theconcentration of C1q, which was reduced at each round with a final round14 C1q concentration of 136 pM. Background binding of RNA tonitrocellulose filters remained low throughout the SELEX procedure, inpart because RNA was pre-adsorbed with nitrocellulose filters. Thebinding of pool RNA to C1q improved at each round. The evolved round 14pool 2′-F RNA bound C1q with a K_(d) of 2 nM, yielding an overallimprovement in binding K_(d) of 1-3000-fold.

[0128] Bulk RNA was then cloned for sequence determination andevaluation of binding. Through comparison of binding at 0.1 and 0.5 nMC1q, individual clones were ranked for binding affinity. Sequences of2′-F RNA ligands are shown in Table 6 (SEQ ID NOS:84-155). The 2′-F-RNAsequences are not easily grouped into families, but these sequences areG-rich and are similar but not homologous with the 2′-NH₂ RNA sequencesdescribed in Example 2.

Example 7

[0129] Hemolytic Inhibition for 2′-F RNA Ligands to C5

[0130] The 2′-F RNA Nucleic Acid Ligands to C5 (Example 4) were assayedfor hemolytic inhibition by including dilutions in a standard assay forhuman serum lysis of antibody-coated sheep erythrocytes. Sheep cellswere mixed with a 1:40 dilution of serum containing Nucleic Acid Ligandor buffer, and incubated for 30 minutes at 37° C. After quenching withcold EDTA buffer, the samples were spun and supernatants read at OD 412nm. Group I Nucleic Acid Ligands inhibited almost to background at 1 μM,with a K_(i) of 60-100 nM. The results are shown in FIG. 1. The resultsof the hemolysis inhibition assay suggested that 2′-F RNA Nucleic AcidLigands to C5 target a specific site on C5, where they block interactionof C5 with the Complement C5 convertase. These results also confirmedthat the 2′-F RNA Nucleic Acid Ligands are stable in serum.

Example 8

[0131] Inhibition of C5a Release

[0132] Nucleic Acid Ligand-C5 interaction that inhibits cleavage of C5would prevent formation of the C5b and MAC assembly. Inhibition of C5cleavage should also inhibit C5a release, and this was shown in thefollowing experiment with clone C6 (SEQ ID NO:51) (Example 4). For thisexperiment, dilutions of clone C6 were incubated with whole human serumin GVB⁺⁺ (veronal-buffered saline containing calcium, magnesium and 1%gelatin) plus addition of zymosan for 30 minutes at 37° C. The sampleswere then quenched with EDTA-buffer and spun, and supernatants wereassayed for C5a by radioimmunoassay (RIA) (Wagner and Hugli (1984) Anal.Biochem. 136:75). The results showed that clone C6 inhibited C5a releasewith a K_(i) of approximately 100 nM (FIG. 2), whereas control randompool RNA gave no inhibition (data not shown). This assay alsodemonstrated the serum stability of clone C6.

Example 9

[0133] Boundaries of Clone C6

[0134] Clone C6 (SEQ ID NO:51) (Example 4) was selected fordetermination of a minimal binding sequence. This was done in thefollowing two ways.

[0135] 1) The minimal RNA sequences (5′ and 3′ boundaries) required forbinding of clone C6 to C5 were determined by partially hydrolyzing cloneC6 and determining protein binding (Green et al. (1995) Chem. Biol.2:683). Briefly, clone C6 was synthesized as either 5′-[³²p]-kinaselabeled (to determine the 3′ boundary) or 3′-[³²P]-pCp labeled (todetermine the 5′ boundary) and the oligonucleotides were purified. Thenthe oligonucleotides were subjected to alkaline hydrolysis, whichcleaves oligonucleotides from the 3′ end to purine bases. The partiallyhydrolyzed RNA was then incubated with C5, and RNA which bound to the C5protein was partitioned on nitrocellulose and eluted from the protein.The partitioned RNA together with an RNA ladder were run on an 8%acrylamide/7M urea sequencing gel. The boundary where removal of onemore base would reduce or eliminate binding was determined by comparisonof selected RNA (RNA which bound to C5) versus non-selected RNA (RNAwhich did not bind to C5).

[0136] The labeled RNA was also digested with T₁ nuclease (which cleavesoligonucleotides from the 3′ end to A residues), incubated with C5 andpartitioned as above, for a second ladder. FIG. 3A shows the results ofthe digestion of the 5′-kinase-labeled RNA. In this figure, the3′-sequence (5′-end labeled) is aligned with the alkaline hydrolysisladder. On the left is the T₁ ladder and on the right are RNA selectedwith 5× and 1× concentrations of C5. The boundary where removal of abase eliminates binding is shown by the arrow. The asterisk shows a Gwhich is hypersensitive to T₁. Other G nucleotides in the minimalsequences are protected from T₁ digestion. FIG. 3B shows the results ofthe 3′-pCp-ligated RNA. In this figure, the 5′-sequence (3′-end-labled)is aligned with the alkaline hydrolysis ladder. The T₁ and proteinlanes, boundary and hypersensitive G nucleotides are as described forFIG. 3A.

[0137] 2) In a second experiment, the results obtained from the boundaryexperiments described above were used to construct synthetic truncatedNucleic Acid Ligands to C5. Several truncates between 34 and 42nucleotides were synthesized by removing residues at both ends of cloneC6 (SEQ ID NO:51), and assayed for C5 binding (Table 8). The shortestoligonucleotide which bound to C5 was a 38 mer (SEQ ID NO:160), whichconfirms the boundary gel and which provides a preliminary structure forfurther Nucleic Acid Ligand development. In the minimal 38 mer sequence,30 bases originated from the random region and eight bases were from the5′ fixed region of clone C6. Removing a base from both 5′ and 3′ ends ofthe 38 mer to produce a 36 mer (SEQ ID NO:161) reduced the binding. A 34mer (SEQ ID NO:162) did not bind. Other truncated oligonucleotides withinternal deletions also failed to bind.

Example 10

[0138] Biased SELEX

[0139] A biased SELEX experiment was performed to improve Nucleic AcidLigand affinity and to further define the structure. The sequence of the42 mer truncate (SEQ ID NO:75) from Example 9 (Table 8)was used as atemplate for the Biased SELEX experiment. A synthetic templatecomprising a 42N random region flanked by new n8 fixed regions (Table10; SEQ ID NO:163) was constructed and synthesized (Oligos, Etc., CT),where the random region was biased toward the 42 mer truncate of cloneC6 from the first SELEX experiment. A 42 mer random region was chosenrather than the minimal 38 mer sequence, as the four extra basesextended a terminal helix. While not wishing to be bound by any theory,the inventors believed that although these four extra bases were notessential for binding, a longer helix was thought desirable to aid inselecting the Nucleic Acid Ligand structure and in minimizing thepossible use of fixed regions in the newly selected Nucleic Acid Ligandstructure. Each base in the random region was synthesized to contain0.67 mole fraction of the base corresponding to the base in the 42 mersequence and 0.125 mole fraction of each of the other three bases. TheBiased SELEX experiment was performed as described for the standardSELEX experiment in Example 1. PCR amplification was performed usingprimers shown in Table 1 (SEQ ID NOS:158-159).

[0140] The Biased SELEX experiment was performed with native C5 proteinsince clone C6 already inhibits hemolysis and trypsin treatment is notrequired for binding. The binding of the starting RNA pool to C5 wasvery low, so the protein and RNA concentrations were started at 2.6 μMand 7.1 μM, respectively, similar to the first SELEX experiment. Thebinding rapidly improved at round three. RNA and protein concentrationswere gradually reduced at each subsequent round to final concentrationsby round nine of 62.5 pM and 31 pM, respectively. The binding of the RNApool to C5 was approximately 5 nM (Table 9), as compared toapproximately 100 M for the RNA pool from the first SELEX experiment.Some of the improvements in the affinity of the pool results fromabsence of lower affinity ligands, size mutants and background binders,which were not allowed to build up to appreciable concentrations duringthis more rapid SELEX experiment.

[0141] The RNA pool after eight rounds of the Biased SELEX process wasimproved by 20-50 fold over the round twelve pool from the first SELEXexperiment. The overall improvement in K_(d) from the random pool topool from eight rounds of the Biased SELEX process is estimated to begreater than 10⁵-fold. The isolated and cloned sequences from the BiasedSELEX experiment are shown in Table 10 (SEQ ID NOS:164-189). In thesequences shown in Table 10, the two base-pair stem which is dispensablefor binding is separated from the minimal 38 mer sequence. These basesshow no selective pressure except to maintain the stem. None of thesequences exactly match the original template sequence.

[0142] Clones from the Biased SELEX experiment were assayed andrepresentative binding affinities are shown in Table 11. Most clonesbound with a K_(d) between 10 and 20 nM and are higher affinity bindingligands than the template (SEQ ID NO:163). One of the clones, YL-13 (SEQID NO:175), bound approximately five-fold higher affinity than otherclones from the Biased SELEX experiment and approximately 10-fold higheraffinity than clone C6 (SEQ ID NO:51). None of Nucleic Acid Ligandsequences exactly matched the sequence used for the template in theBiased SELEX experiment. Some bases substitutions are unique to thisBiased SELEX experiment sequence set and might account for increasedNucleic Acid Ligand affinity.

Example 11

[0143] 2′-O-Methyl Substitution for Nuclease Protection

[0144] To further stabilize the Nucleic Acid Ligand, positions where2′-OH-purine nucleotides could be substituted with nuclease-resistant2′-O-methyl nucleosides were determined. An assay for simultaneouslytesting several positions for 2′-O-methyl interference was usedfollowing the method described in Green et al. (1995) Chem. Biol. 2:683.

[0145] In the 2′-O-methyl interference assay, three sets ofoligonucleotides based on a 38 mer truncate of sequence YL-13 (SEQ IDNO:175) from the Biased SELEX experiment were synthesized. These sets ofsequences, indicated as M3010 (SEQ ID NO:190), M3020 (SEQ ID NO:191) andM3030 (SEQ ID NO:192) in Table 12 were synthesized on an automated RNAsynthesizer in a manner wherein each of the nucleotides indicated bybold underline in Table 12 were synthesized 50% as a 2′-OH-nucleotideand 50% as a 2′-OMe-substituted nucleotide. This resulted in a mixtureof 2⁵ or 32 different sequences for each of sets M3010, M3020 and M3030.

[0146] The partially substituted 2′-OMe oligonucleotides were5′-[³²P]-kinase-labeled. The oligonucleotides were selected at 100 nMand 10 nM C5 and the binding to protein was greater than 10-fold overbackground filter binding. The oligonucleotides were eluted from theprotein, alkaline hydrolyzed and then run on a 20% acrylamide/7 Murea/TBE sequencing gel. On adjacent tracks were run oligonucleotidesnot selected with C5. Band intensities were quantitated with on anInstantImager (Packard, Meriden, Conn.). When these oligonucleotideswere separated on an acrylamide gel the mixed OH:OMe positions showed upat 50% intensity of a full 2′-OH position, because the 2′-OMe isresistant to hydrolysis. 2′-F pyrimidines are also resistant and do notshow on the gel.

[0147] For each position, the ratio of (the intensity of the bandsselected by protein binding)/(band intensity for oligonucleotide notselected to protein) was calculated. These ratios were plotted versusnucleotide position and a linear fit determined (FIG. 4, open circles).The same calculation was made for mixed 2′-OH/2′-OMe oligonucleotides,and these ratios were compared with previously determined curve (FIG. 4,closed circles). Where 2′-OMe substitution did not interfere withbinding the ratio was within one standard deviation of the 2′-OH ratio.However, where 2′-OMe substitution interfered with binding, the bindingpreference for 2′-OH purine increased the ratio. Two nucleotides atpositions 16 and 32 were determined to require 2′-OH nucleotides.Separately, residue g5 was determined independently to require 2′-OH andresidue G20 was determined to allow 2′-OMe substitution, and these wereused to normalize lanes. These results were confirmed by synthesis andassay of 2′-OMe substituted oligonucleotides. The obligate 2′-OHpositions are in one of two bulges, or in the loop in the putativefolding structure, suggesting these features are involved in the proteininteraction. Once the permissible 2′-OMe positions were determined,substituted oligonucleotides were synthesized and relative bindingaffinities were measured.

Example 12

[0148] Human C5 Nucleic Acid Ligand Structure

[0149] The putative folding and base-pairing, based on truncationexperiments, nuclease sensitivity, base substitution patterns from theBiased SELEX experiment, and 2′-OMe substitutions, for the 38 mertruncate of clone C6 together with alternative bases is shown in FIG.5A. The basic sequences is the 38 mer truncate (SEQ ID NO:160). Inparentheses are variants from the first SELEX experiment. In bracketsare variants from the Biased SELEX experiment. Lower case bases arederived from the 5′-n7 fixed region from the first SELEX experiment.Upper case bases are derived from the original random region.

[0150] The stem-loop structure has between 12 and 14 base-pairs: a) theproposed 5′, 3′-terminal base pairs (c1-a3, and U36-G38); b) stem-loopbase-pairs (U11-U14 and G24-A27) are supported by covariant changesduring the Biased SELEX procedure; and c) the middle stem (g7-C10 andG28-C34), which is generally conserved, U9-A32 which is invariant andg8-C33 conserved during the Biased SELEX procedure. The u4→c4 changeimproves binding, and this change is found in all clones from the BiasedSELEX experiment, and G29, A29 variants are found only in clones fromthe Biased SELEX experiment.

[0151] The UUU bulge is generally conserved. One original sequencecontained two U bases, with no reduction in binding, and two NucleicAcid Ligands with a single base substitution were found during BiasedSELEX experiment. The C10-G28 base-pair following the UUU-bulge isconserved. This region with a conserved bulge and stem is likelyinvolved in protein interaction. The stem-loop G15 to U23 is highlyconserved, except for bases 19.

[0152] The 2′-OMe substitution pattern is consistent with this structure(SEQ ID NO:193; FIG. 5B). Positions where 2′-OMe substitutions can bemade are shown in bold. The three positions which must be 2′-OH areshown as underlined. The obligate 2′-OH bases at g5, G17 and A32 are inbulge or loop regions which might form unique three-dimensionalstructures required for protein binding. Allowed positions for 2′-OMesubstitution occur in stem regions where a standard helical structure ismore likely.

Example 13

[0153] Hemolytic Assay of 2′-OMe-Substitued Nucleic Acid Ligands toHuman C5

[0154] Three oligonucleotides were synthesized based on clone YL-13 fromthe Biased SELEX experiment to compare the effect of 2′-OMe substitutionon hemolytic inhibition: (1) a 38 mer truncate B2010 (SEQ ID NO:194), inwhich all of the nucleotides were 2′-OH; (2) a 38 mer in which onenucleotide (position 20) was a 2′-OMe-G (B2070; SEQ ID NO:195); and (3)a 38 mer in which the maximum number of allowable positions (positions2, 7, 8, 13, 14, 15, 20, 21, 22, 26, 27, 28, 36 and 38) were synthesizedas 2′-OMe-G and 2′-OMe-A (M6040; SEQ ID NO:196) as shown in Table 13.These were assayed in the hemolytic assay as described in Example 7. Theresults are shown in FIG. 6. As shown in FIG. 6, the K_(i) decreasedwith increased 2′-O-Me substitution. The K_(d) was marginally better(data not shown). This experiment showed that nucleic acid ligandstability is increased with 2′-OMe substitution, and that long term invivo inhibition of the complement system is feasible. TABLE 1 SEQ ID NO.Synthetic DNA Template: 1 and 1565′-TAATACGACTCACTATAGGGAGGACGATGCGG-[N]_(30 or 50-) CAGACGACTCGCCCGA-3′Starting random sequence RNA pool: 2 and 1575′-GGGAGGACGAUGCGG-[N]_(30 or 50)-CAGACGACUCGCCCGA-3′ Primer Set forStandard SELEX: 3 5′-PRIMER: 5′-TAATACGACTCACTATAGGGAGGACGATGCGG-3′ 43′-PRIMER: 5′-TCGGGCGAGTCGTCTG-3′ Primer Set for Biased SELEX: 1585′-PRIMER: 5′-TAATACGACTCACTATAGGGAGATAAGAATAAACGCTCAA-3′ 159 3′ PRIMER:5′ GCCTGTTGTGAGCCTCCTGTCGAA-3′

[0155] TABLE 2 2′-NH₂ RNA Ligands of Complement System Protein Clq* SEQID Clone No. NO: Kd(nM) Family 1 3gggaggacgaugcggGAGGAGUGGAGGUAAACAAUAGGUCGGUAGCGACUCCCACUAACAGGCCUcagacgacucgcccga5 12gggaggacgaugcggGUGGAGUGGAGGUAAACAAUAGGUCGGUAGCGACUCCCAGUAACGGCCUcagacgacucgcccga6 23cgggaggacgaugcaaGUGGAGUGGAGGUAUAACGGCCGGUAGGCAUCCCACUCGGGCCUAGCUcagacgacucgcccga7 30gggaggacgaugcggGUGGAGUGGGGAUCAUACGGCUGGUAGCACGAGCUCCCUAACAGCGGUcagacgacucgcccga8 36gggaggacgaugcggGAGGAGUGGAGGUAAACAAUAGGCCGGUAGCGACUCCCACUAACAGCCUcagacgacucgcccga9 0.29 45gggaggacgaugcggUGGAGUGGAGGUAUACCGGCCGGUAGCGCAUCCCACUCGGGUCUGUGCUcagacgacucgcccga10 1.38 47gggaggacgaugcggGUGGAGCGGAGGUUUAUACGGCUGGUAGCUCGAGCUCCCUAACACGCGGUagacgacucgcccga11 50gggaggacgaugcggGUGGAGUGGAGGUAUAACGGCCGGUAGCGCAUCCCACUCGGGUCUGUGCUagacgacucgcccga12 0.979 78gggaggacgaugcggGUGGAGUGGAGGGUAAACAAUGGCUGGUGGCAUUCGGAAUCUCCCAACGUagacgacucgcccga13 Family 2 33gggaggacgaugcggGUUGCUGGUAGCCUGAUGUGGGUGGAGUGAGUGGAGGGUUGAAAAAUGcagacgacucgcccga14 3.85 40gggaggacgaugcggCUGGUAGCAUGUGCAUUGAUGGGAGGAGUGGAGGUCACCGUCAACCGUcagacgacucgcccga15 43gggaggacgaugcggUUUCUCGGCCAGUAGUUUGCGGGUGGAGUGGAGGUAUAUCUGCGUCCUCGcagacgacucgcccga16 Family 3 14gggaggacgaugcggCACCUCACCUCCAUAUUGCCGc3UUAUCGCGUAGGGUGAGCCCAGACACGAcagacgacucgcccga17 2.4 23gggaggacgaugcggCACUCACCUUCAUAUUGGCCGCCAUCCCCAGGGUUGAGCCCAGACACAGcagacgacucgcccga18 23 Family 4 22gggaggacgaugcggGCAUAGUGGGCAUCCCAGGGUUGCCUAACGGCAUCCGGGGUUGUUAUUGGcagacgacucgcccga19 67gggaggacgaugcggCAGACGACUCGCCCGAGGGGAUCCCCCGGGCCUGCAGGAAUUCGAUAUcagacgacucgcccga20

[0156] TABLE 3 2′-F RNA Ligands of Complement System Protein of HumanC3* SEQ Clone No. ID NO: C3c 10 gggaggacgaugcggAACUCAAUGGGCCUACUUUUUCCGUGGUCCU cagacgacucgcccga 21 C3C 16gggaggacgaugcgg AACUCAAUGGGCCUACUUUUCCGUGGUCCU cagacgacucgcccga 22 C3C186 gggaggacgaugcgg AACUCAAUGGGCCGACUUUUUCCGUGUCCU cagacgacucgcccg 23C3C 162 gggaggacgaugcgg AACUCAAUGGGCCGACUUUCCGUGGUCCU cagacgacucgcccga24 C3C 141 gggaggacgaugcgg AACUCAAUGGGCNUACUUUUCCGUGGUCCUcagacgacucgcccga 25 C3c 32 gggaggacgaugcggAACUCAAUGGGCCGACUUUUCCGUGGUCCU cagacgacucgcccga 26 27C3B143gggaggacgaugcgg AACUCAAUGGGCCGACUUUUCCGUGGUCCU cagacgacugcccga 2730C3B149 gggaggacgaugcgg ACGCAGGGGAUGCUCACUUUGACUUUUAGGC cagacgacucgcccg28 c3a 29c gggaggacgaugcgg ACUCGGCAUUCACUAACUUUUGCGCUCGUcagacgacucgcccga 29 C3B 25 gggaggacgaugcggAUAACGAUUCGGCAUUCACUAACUUCUCGU cagacgacucgcccga 30 C3c 3 gggaggacgaugcggAUGACGAUUCGGCAUUCACUAACUUCUCGU cagacacucgcccga 31 C3C 155gggaggacgaugcgg AUGACGAUUCGGCAUUCACUAACUUCUCAU cagacgacucgcccga 32 C3C109 gggaggacgaugcgg AUGACGAUUCGGCAUUCACUAACUUCUACU cagacgacucgcccga 33C3-A 18c gggaggacgaugcgg AUCUGAGCCUAAAGUCAUUGUGAUCAUCCU cagacgacucgcccga34 C3c 35 gggaggacgaugcggg CGUUGGCGAUUCCUAAGUGUCGUUCUCGUcagacgacucgcccga 35 C3B 41 gggaggacgaugcgg CGUCUCGAGCUCUAUGCGUCCUCUGUGGUcagacgacucgcccga 36 C3B 108 gggaggacgaugcggCGUCACGAGCUUUAUGCGUUCUCUGUGGU cagacgacucgcccga 37 C3c 77 gggaggacgaugcggCUUAAAGUUGUUUAUGAUCAUUCCGUACGU cagacgacucgcccga 38 C3B 102gggaggacgaugcgg GCGUUGGCGAUUGGUAAGUGUCGUUCUCGU cagacgacucgcccga 39 c3a9c gggaggacgaugcgg GCGUCUCGAGCUUUAUGCGUUCUCUGUGGU cagacgacucgcccga 40C3B 138 gggaggacgaugcgg GCGUCUCGAGCUCUAUGCGUUCUCUGUGGU cagacgacucgcccga41 c3-8c ggaggacgaugcgg GGCCUAAAGUCAAGUGAUCAUCCCCUGCGU cagacganucgcccga42 C3-230 gggaggacgaugcgg GUGGCGAUUCCAAGUCUUCCGUGAACAUGGUcagacgacucgcccg 43 C3c 36 gggaggacgaugcggGUGACUCGAUAUCUUCCAAUCUGUACAUGGU cagacgacucncccga 44 188 gggaggacgaugcggUGGCGAUUCCAAGUCUUCCGTGAACATGGT cagacgacucgcccga 45 C3B 23gggaggacgaugcgg TGGCGATTCCAAGTCTTCCGTGAACAT cagacgacucgcccga 46

[0157] TABLE 4 2′-F RNA Ligands of Complement System Protein Human C5*SEQ Clone No: ID NO: Group I E5c/E11 gggaggacgaugcggUCCGGCGCGCUGAGUGCCGGUUAUCCUCGU cagacgacucgcccga 47 A6 gggaggacgaugcggUCCGGCGCGCUGAGUGCCGGUUUAUCCUCGU cagacgacucgcccga 48 F8 gggaggacgaugcggUCUCAUGCGCCGAGUGUGAGUUUACCUUCGU cagacgacucgcccga 49 K7 gggaggacgaugcggUCUCAUGCGUCGAGUGUGAGUUUAACUGCGU cagacgacucgcccga 50 C6 gggaggacgaugcggUCUCAUGCGUCGAGUGUGAGUUUACCUUCGU cagacgacucgcccga 51 G7 gggaggacgaugcggUCUGCUACGCUGAGUGGCUGUUUACCUUCGU cagacgacucgcccga 52 H1 gggaggacgaugcggUCGGAUGCGCCGAGUCUCCGUUUACCUUCGU cagacgacucgcccga 53 Group II F11gggaggacgaugcgg UGAGCGCGUAUAGCGGUUUCGAUAGAGCUGCGU cagacgacucgcccga 54 H2gggaggacgaugcgg UGAGCGCGUAUAGCGGUUUCGAUAGAGCCU cagacgacucgcccga 55 H6gggaggacgaugcgg UGAGCGUGGCAAACGGUUUCGAUAGAGCCU cagacgacucgcccga 56 H8gggaggacgaugcgg UGAGCGUGUAAAACGGUUUCGAUAGAGCCU cagacgacucgcccga 57 C9gggaggacgaugcgg UGAGCGUGUAAAACGGUUUCGAUAGAGCCU cagacgacucgcccga 58 C12gggaggacgaugcgg UGGGCGUCAGCAUUUCGAUCUUCGGCACCU cagacgacucgcccga 59 G9gggaggacgaugcgg GAGUUGUUCGGCAUUUAGAUCUCCGCUCCCU cagacgacucgcccga 60 F7gggaggacgaugcgg GCAAAGUUCGGCAUUCAGAUCUCCAUGCCCU cagacgacucgcccga 61 E9cgggaggacgaugcgg GGCUUCUCACAUAUUCUUCUCUUUCCCCGU cagacgacucgcccga 62 E4cgggaggaggaucgg UGUUCAGCAUUCAGAUCUU cagacgacucgcccga 63 G3gggaggacgaugcgg UGUUCAGCAUUCAGN/AUCUUCACGUGUCGU cagacgacucgcccga 64 F6gggaggacgaugcgg UGUUCACCAUUCAGAUCUUCACGUGUCGU cagacgacucgcccga 65 D9gggaggacgaugc UGUUCAGCAUUCAGAUCUUCACGUGUGU cagacgacucgcccga 66 F4gggaggacgaugcgg UUUCGAUAGAGACUUACAGUUGAGCGCGGU cagacgacucgcccga 67 D3gggaggacgaugcgg UUUGUGAUUUGGAAGUGGGGGGGAUAGGGU cagacgacucgcccga 68 F9gggaggacgaugcgg UGAGCGUGGCAAACGGUUUCGAUAGAGCCU cagacgacucgcccga 69 J1cggagggcgaugg GGUGAGCGUGUAAAAGGUUGCGAUAGAGCCU cagacgacucgcccga 70 D6gggaggacgaugcgg GUAUCUUAUCUUGUUUUCGUUUUUCUGCCCU cagacgaucgcccga 71 E8xgggaggacgaugcgg AGGGUUCUUUUCAUCUUCUUUCUUUCCCCU cagacgacucgcccga 72 H11gggaggacgaugcgg ACGAAGAAGGUGGUGGAGGAGUUUCGUGCU cagacgacucgcccga 73 G10gggaggacgaugcgg ACGAAGAAGGGGGUGGAGGAGUUUCGUGCU cagacuacucgcccga 74

[0158] TABLE 5 Rat C5 2′F- RNA seguences* SEQ Clone No: ID NO: Family IRtC5-116 gggaggacgaugcgg CGAUUACUGGGACGGACUCGCGAUGUGAGCCcagacgacucgcccga 76 RtC5-39 gggaggacgaugcggCGAUUACUGGGACAGACUCGCGAUGUGAGCU cagacgacucgcccga 77 RtC5-69gggaggacgaugcgg CGACUACUGGGAAGGGUCGCGGUGAGCC cagacgacucgcccga 78 RtC5-95gggaggacgaugcgg CGAUUACUGGGACAGACUCGCGAUGUGAGCU cagacgacucgcccga 79RtC5-146 gggaggacgaugcgg CGACUACUGGGAGAGUACGCGAUGUGUGCC cagacgacucgcccga80 Family II RtC5-168 gggaggacgaugcgg GUCCUCGGGGAAAAUUUCGCGACGUGAACCUcagacgacucgcccga 81 Family III RtC5-74 gggaggacgaugcggCUUCUGAAGAUUAUUUCGCGAUGUQAACUUCAGACCCCU cagacgacucgcccga 82 RtC5-100gggaggacgaugcgg CUUCUGAAGAUUAUUUCGCGAUGUGAACUCCAGACCCCU cagacgacucgcccga83

[0159] TABLE 6 2′-F RNA Ligands of Complement System Protein Clq* SEQClone No: ID NO: c1qrdl7-33c gggaggacgaugcggAAAGUGGAAGUGAAUGGCCGACUUGUCUGGU cagacgacucgcccga 84 C1B100gggaggacgaugcgg AAACCAAAUCGUCGAUCUUUCCACCGUCGU cagacgacucgcecga 85c1q-a8c gggaggacgaugcgg AACACGAAACGGAGGUUGACUCGAUCUGGC cagacgacucgcccga86 C1q5 cggaggacgaugcgg AACACGGAAGACAGUGCGACUCGAUCUGGUcagacgacucgcccga87 32.C1B76 cgggaggacgaugcgg AACAAGGACAAAAGUGCGAUUCUGUCUGGcagacgacucgcccg 88 c110c gggaggacgaugcgg AACAGACGACUCGCGCAACUACUCUGACGUcagacgacucgcccga 89 C1B121c gggaggacgaugcggAACAGGUAGUUGGGUGACUCUGUGUGACCU cagacgacucgcccga 90 C1q11ccggaggacgaugcgg AACCAAAUCGUCGAUCUUUCCACCGCUCGU cagacgacucgcccga 91 C15ccgggaggacgaugcgg AACCGCUAUUGAAUGUCACUGCUUCGUGCU cagacgacucgcccga 92C1Q-A24′c cgggaggacgaugcgg AACCGCAUGAGUUAGCCUGGCUCG 93 C1Q-A5′cgggaggacgaugcgg AACCCAAUCGUCUAAUUCGCUGCUCAUCGUcagacgacucgcccga 94 C121cgggaggacgaugcgg AACUCAAUGGGCCUACULTUUCCGUGGUCCUcagacgacucgcccga 95c1q-a2C gggaggacgaugcgg AAGCGGUGAGUCGUGGCUUUCUCCUCGAUCCUCGUcagacgacucgcccga 96 c1q-a12C gggaggacgaugcggAAGGAUGACGAGGUGGUUGGGGUUUGUGCUcagacgacucgcccga 97 c1qrd17-43cgggaggacgaugcgg ACAAGACGAGAACGGGGGGAGCUACCUGGC cagacgacucgcccga 98CIQ-A7′C gggaggacgaugcgg AGACACUAAACAAAUUGGCGACCUGACCGU cagacgacucgcccga99 03.C1Q.137c gggaggacgaugcgg AGACGCUCAGACGACUCGCCCGACCACGGAUGCGACCUcagacgacucgcccga 100 14.C1Q156c gggaggacgaugcggAGAUGGAUGGAAGUGCUAGUCUUCUGGGGU cagacgacucgccc 101 C1B119cgggaggacgaugcgg AGAUGGAUGGAAGUGCUAGUCUUUCUGGGGU cagacgacucgcccga 102C1Q-A28′C gggaggacgaugcqg AGCAGUUGAAAGACGUGCGUUUCGUUUGGUcagacgacucgcccga 103 15.C1Q.157c gggaggacgaugcggAGCACAAUUUUUUCCUUUUCUUUUCGUCCACGUGCU cagacgacucgcccga 104 44c1qb60cgggaggacgaugcgg AGCUGAUGAAGAUCAUCUCUGACCCCU cagacgacucgcccga 10506.C1Q.143c gggaggacgaugcgg AGCUGAAAGCGAAGUGCGAGGUCUUUGGUCcagacgacucgcccga 106 C1q4c ggaggacgaugcgg AGCGAAAGUGCGAGUGAUUGACCAGGUGCUcagacgacucgcccga 107 c1qrd17-52c gggaggacgaugcggAGCGUGAGAACAGUUGCGAGAUUGCCUGGU cagacgacucgcccga 108 C111cgggaggacgaugcgg AGGAGAGUGUGGUGAGGGUCGUUUUGAGGGU cagacgacucgcccga 10944c1Qb60c gggaggacgaugcgg AGGAGCUGAUGAAGAUGAUCUCUGACCCCUcagacgacucgcccga 110 24c1qb51C gggaggacgaugcggAGUUCCCAGCCGCCUUGAUUUCUCCGUGGU cagacgacucgcccga 111 31c1qb16cgggaggacgaugcgg AUAAGUGCGAGUGUAUGAGGUGCGUGUGGU cagacgacucgcccga 11228c1Qb20c gggaggacgaugcgg AUCUGAGGAGCUCUUCGUCGUGCUGAGGGUcagacgacucgcccga 113 c1qrd17-61c gggaggacgaugcggAUCCGAAUCUUCCUUACACGUCCUGCUCGU cagacgacucgcccga 114 C1q17cggaggacgaugcgg AUCCGCAAACCGACAGCUCGAGUUCCGCCU cagacgacucgcccga 11534c1qb27c gggaggacgaugcgg AUGGUACUUUAGUCUUCCUUGAUUCCGCCUcagacgacucgcccga 116 C1q7c cggaggacgaugcggAUGAUGACUGAACGUGCGACUCGACCUGGC cagacgacucgcccga 117 C1q7c ggaggacgaugcggAUGAGGAGGAAGAGUCUGAGGUGCUGGGGU cagacgacucgcccga 118 C1Q-A22′Cgggaggacgaugcgg AUUUCGGUCGACUAAAUAGGGGUGGCUCGU cagacgacucgcccga 119C122c gggaggacgaugcgg CAAGAGGUCAGACGACUGCCCCGAGUCCUCCCCCGGUcagacgacucgcccga 120 C115c gggaggacgaugcgg CAGUGAAAGGCGAGUUUUCUCCUCUCCCUcagacgacucgcccga 121 09.C1Q.149c gggaggacgaugcggCAUCGUUCAGGAGAAUCCACUUCGCCUCGU cagacgacucgcccga 122 04.C1Q.138cgggaggacgaugcgg CAUCUUCCUUGUUCUUCCAACCCUCCUCCU cagacgacucgcccga 123C1Q-A4′C gggaggacgaugcgg CAUCGUAAACAAUUUGUUCCAUCUCCGCCU cagacgacucgcccga124 c1qrdl7-64c gggaggacgaugcgg CAUUGUCCAAGUUUAGCUGUCCGUGCUCGUcagacgacucgcccga 125 46C1Qb64c gggaggacgaugcggCAUACUCCGGAUACUAGUCACCAGCCUCGU agacgacucgcccga 126 Cliq6cgggaggacgaugcgg CCGUCUCGAUCCUUCUAUGCCUUCGCUCGU cagacgacucgcccga 12723C1Qb4x gggaggacgaugcgg CGGGAAGUUUGAGGUGUANUACCUGUUGUCUGGUcagacgacucgcccga 128 c1qrd17-63c gggaggacgaugcggCUCAACUCUCCCACAGACGACUCGCCCGGGCCUCCU cagacgacucgcccga 129 c1qrd17-47cgggaggacgaugcgg GACUCCUCGACCGACUCGACCGGCUCGU cagacgacucgccga 130 C1g9cggaggacgaugcgg GAACCAAAUCGUCGAUCUUUCCACCGCUCGU cagacgacucgcccga 131C1qrd-A63c10 cggaggacgaugcgg GACCACCUCGAUCCUCAGCGCCAUUGCCCUcagacgacucgcccga 132 C119c gggaggacgaugcgg GAAGUGGAAGGGUAGUUGUGUGACCUcagacgacucgcccga 133 c1qrd17-42c cggaggacgaugeggGCAAACUTUUUCCUUUUCCCUUUAUCUUCCUUGCCCU cagacgacucgcccga 134 30c1Q24cgggaggacgaugcgg GGCCGACGAUUCACCAAUGUUCUCUCUGGU cagacgacucgcccga 135C1q10c ggaggacgaugcgg GGUUCCUCAAUCACGAUCUCCAUUCCGCUCGU cagacgacucgcccag136 C1q20c ggaggacgaugcgg GUCGACAUUGAAGCUGCUCUGCCUUGAUCCUcagacgacucgcccga 137 08.C1Q.147c gggaggacgaugcggUCCAAUUCGUUCUCAUGCCUUUCCGCUCGU cagacgacucgcccga 138 11.C1Q.152cgggaggacgaugcgg UCCGCAACUUUAGCACUCACUGCCUCGU cagacgacucgcccga 13926c1Qb4c gggaggacgaugcgg UCCACAUCGAAUUUUCUGUCCGUUCGU cagacgacucgeccga140 C1B115c gggaggacgaugcgg UCGAUGUUCUUCCUCACCACUGCUCGUCGCCUcagacgacucgcccga 141 33c1Q26c gggaggacgaugcggUCGAGCUGAGAGGGGCUACUUGUUCUGGUCA cagacgacucgcccga 142 01.C1Q.135cgggaggacgaugcgg UGGAAGCGAAUGGGCUAGGGUGGGCUGACCUC cagacgacucgcccga 14347c1qb65 cgggaggacgaugcgg UGGACUUCUUUUCCUCUUUCCUCCUUCCGCCGGUcagacgacucgcccga 144 C1q14c ggaggacgaugcggUUCCAAAUCGUCUAAGCAUCCCUCGCUCGU cagacgacucgcccag 145 c1qrd17-53cgggaggacgaugcgg UUCCACAUCGCAAUUUUCUGUCCGUGCUCGU cagacgacucgcccga 146c1q-a6C gggaggacgaugcgg UUCCACAUCGAAUUUUCUGUCCGUGUCGU cagacgacucgcccga147 C1B114 cgggaggacgaugcgg UUCCGAUCGACUCCACAUACAUCUGCUCGUcagacgacucgcccga 148 c1qrd17-56c gggaggacgaugcggUUCCGACAUCGAUGUUGCUCUUCGCCUCGU cagacgacucgcccga 149 05.C1Q.142cgggaggacgaugcgg UUCCGAAGUUCUUCCCCCGAGCCUUCCCCCUC cagacgacucgcccga 15030c1q24 cgggaggacgaugcgg UUCCGACGAUUCUCCAAUGUUCUCUCUGGU cagacgacucgcccga151 38c1qb45c gggaggacgaugcgg UUCCGACGAUUCUCCAAUCUUCUCUCUGGUcagacgacucgcccga 152 10.C1Q151c gggaggacgaugcggUUCCGCAAGUUUAGACACUCACUGCCUCGU cagacgacucgcccga 153 C113xgggaggacgaugcgg UUCCGCAAAGUAGAUAUNUCAUCCGCACCU cagacgacucgcccga 15410.C1B.134c gggaggacgaugcgg UUGAGUGGACAGUGCGAUUCGUUUUGGGGUcagacgacucgcccga 155

[0160] TABLE 7 Binding affinity of C5 nucleic acid ligands Clone SEQ IDNO Kd (nM) A6 48 35  E11 47 60 E4 63 50 C6 51 30 C9 58 45 G3 64 55 F8 4930

[0161] TABLE 8 Effect of truncation of clone C6 on C5 binding Length KdSEQ ID NOS: Sequence (nts) (nM) *75 gACgAUgCggUCUCAUgCgUCgAgUgUgAgUUUACCUUCg UC 42 160CgAUgCggUCUCAUgCgUCgAgUgUgAgUUUACCUUCg 38 20 161gAUgCggUCUCAUgCgUCgAgUgUgAgUUUACCUUC 36 50 162AUgCggUCUCAUgCgUCgAgUgUgAgUUUACCUU 34 >10⁶

[0162] TABLE 9 Binding of SELEX pools SELEX pool Kd random pool >1 nMFirst SELEX, round 12 100 nM Biased SELEX, round 8 5 nM

[0163] TABLE 10 Clones from Biased SELEX SEQ ID Clone No. NO: templategggagataagaataaacgctcaag GA CGATGCGGTCTCATGCGTCGAGTGTGAGTTTACCTTCG TCttcgacaggaggctcacaacaggc 163 YL-8(10): gggagauaagaauaaacgcucaag UGCGACGCGGUCUCGAGCGCGGAGUUCGAGUUUACCUUCG CA uucgacaggaggcucacaacaggc 164YL-33(2): gggagauaagaauaaacgcucaag CUCGACGCGGUCCCAGGCGUGGAGUCUGGGUUUACCUUCG AG uucgacaggaggcucacaacaggc 165YL-79(3): gggagauaagaauaaacgcucaag AACCACGCGGUCUCAGGCGUAGAGUCUGAGUUUACCUUGG UU uucgacaggaggcucacaacaggc 166YL-1(2): gggagauaagaauaaacgcucaag AACCACGCGGUCUCAGGCGUAGAGUCUGUGUUUACCUUGG UU uucgacaggaggcucacaacaggc 167YL-71: gggagauaagaauaaacgcucaag UGCGACGCGGUCUCGAGCGCGGAGUUCGAGUUCACCUUCG CA uucgacaggaggcucacaacaggc 168YL-39: gggagauaagaauaaacgcucaag CACAACGCGGUCUCAUGCGUCGAGUAUGAGUUUACCUUuG UG uucgacaggaggcucacaacaggc 169YL-60: gggagauaagaauaaacgcucaag GUCCUCGCGGUCUCAUGCGCCGAGUAUGAGUUUACCUAGG AC uucgacaggaggcucacaacaggc 170YL-9: gggagauaagaauaaacgcucaag GU CGUCGCGGUCUGAUGCGCUGAGUAUCAGUUUACCUACGAC uucgacaggaggcucacaacaggc 171 YL-56: gggagauaagaauaaacgcucaag GUACACGCGGUCUGACGCGCUGAGUGUCAGUUUACCUUGU AC uucgacaggaggcucacaacaggc 172YL-63: gggagauaagaauaaacgcucaag AAACCACGCGGUCUCAGGCGCAGAGUCUGAGUUACCUUCGCA uucgacaggaggcucacaacaggc 173 YL-29: gggagauaagaauaaacgcucaag AACCACGCGGUCUCAGGCGCAGAGUCUGAGUUACCUUGG UU uucgacaggaggcucacaacaggc 174YL-13: gggagauaagaauaaacgcucaag GACGCCGCGGUCUCAGGCGCUGAGUCUGAGUUUACCUGCG UC uucgacaggaggcucacaacaggc 175YL-24: gggagauaagaauaaacgcucaag GCUGACGCGGUCUCAGGCGUGGAGUCUGAGUUUACCUUCG GC uucgacaggaggcucacaacaggc 176YL-3: gggagauaagaauaaacgcucaag CA UGACGCGGUCUCAGGCGUGGAGUCUGAGUUUACCUUCGUG uucgacaggaggcucacaacaggc 177 YL-67: gggagauaagaauaaacgcucaag GUCGACGCGGUCUCAGGCGUUGAGUCUGUGUUUACCUUCG AC uucgacaggaggcucacaacaggc 178YL-69: gggagauaagaauaaacgcucaag GUCGACGCGGUCUCAGGCGUUGAGUCUGUGUUUACCUUCG AC uucgacaggaggcucacaacaggc 179YL-81: gggagauaagaauaaacgcucaag GACGCCGCGGUCUCAGGCGUUGAGUCUGAGUUUACCUGCG UC uucgacaggaggcucacaacaggc 180YL-15(7): gggagauaagaauaaacgcucaag GACGACGCGGUCUGAUGCGCUGAGUGUCAGUUUACCUUCG UC uucgacaggaggcucacaacaggc 181YL-84: gggagauaagaauaaacgcucaag AACGACGCGGUCUGAUGCGCUGAGUGUCAGUGUACCUUCG UC uuogacaggaggcucacaacaggc 182YL-4(3): gggagauaagaauaaacgcucaag GUCGACGCGGUCUGAUGCGUAGAGUGUCAGUUUACCUUCG AC uucgacaggaggcucacaacaggc 183YL-51: gggagauaagaauaaacgcucaag GUCGACGCGGUCUGAUGCGUAGAGUGUCAGUUCACCUUCG AC uucgacaggaggcucacaacaggc 184YL-14(2): gggagauaagaauaaacgcucaag UACGACGCGGUCCCGUGCGUGGAGUGCGGGUUUACCUUCG UA uucgacaggaggcucacaacaggc 185YL-23: gggagauaagaauaaacgcucaag GACGACGCGGUCUGAUGCGCAGAGUGUCGGUUUACCUUUG UC uucgacaggaggcucacaacaggc 186YL-59: gggagauaagaauaaacgcucaag GACGACGCNGUCUGAUGCGCAGAGUGUCAGUUUACCUUCG AC uucgacaggaggcucacaacaggc 187YL-91: gggagauaagaauaaacgcucaag GACGACGCGGUCUGAUGCGCAGAGUGUCAGUUUACCUUCG UC uucgacaggaggcucacaacaggc 188YL-50: gggagauaagaauaaacgcucaag GACGACGCGGUCGGAUGCGCAGAGUGUCCGUUUACCUUCG UC uucgacaggaggcucacaacaggc 189

[0164] TABLE 11 Binding affinity of clones from Biased SELEX experimentSEQ ID NO: Clone Kd (nM) 166 YL-79 15 172 YL-56 12 175 YL-13  6 185YL-14 25 163 Template 30

[0165] TABLE 12 Sequences based on YL-13 from Biased SELEX CloneSequence SEQ ID NO: M3010 CGC CGC GGU CUC AGG CGC UGA GUC UGA GUU UACCUG CG 190 M3020 CGC CGC GGU CUC GGG CGC UGA GUC UGA GUU UAC CUG CG 191M3030 CGC CGC GGU CUC AGGCGC UGA GUC UGA GUU UAC CUG CG 192

[0166] TABLE 13 Truncates based on YL-13 for hemolytic assay CloneSequence SEQ ID NO: YL-13t CGC CGC GGU CUC AGG CGC UGA GUC UGA GUU UACCUG CG 194 B2070 CGC CGC GGU CUC AGG CGC UGA GUC UGA GUU UAC CUG CG 195M6040 CGC CGC GGU CUCAGG CGC UGA GUC UGA GUU UAC CUGCG196

[0167]

1 198 1 78 DNA Artificial Sequence unsure (33)..(62) Description ofArtificial Sequence Completely Synthesized Nucleic Acid. N′s at position33-62 are a or c or g or t 1 taatacgact cactataggg aggacgatgc ggnnnnnnnnnnnnnnnnnn nnnnnnnnnn 60 nncagacgac tcgcccga 78 2 61 RNA ArtificialSequence unsure (16)..(55) Description of Artificial Sequence CompletelySynthesized Nucleic Acid. N′s at position 16-55 are a or c or g or u. 2gggaggacga ugcggnnnnn nnnnnnnnnn nnnnnnnnnn nnnnncagac gacucgcccg 60 a61 3 32 DNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 3 taatacgact cactataggg aggacgatgcgg 32 4 16 DNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 4 tcgggcgagt cgtctg 16 5 81 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 5 gggaggacga ugcgggagga guggagguaa acaauaggucgguagcgacu cccacuaaca 60 ggccucagac gacucgcccg a 81 6 80 RNA ArtificialSequence Description of Artificial Sequence Completely SynthesizedNucleic Acid 6 gggaggacga ugcgggugga guggagguaa acaauagguc gguagcgacucccaguaacg 60 gccucagacg acucgcccga 80 7 79 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid 7gggaggacga ugcaagugga guggagguau aacggccggu aggcauccca cucgggccua 60gcucagacga cucgcccga 79 8 79 RNA Artificial Sequence Description ofArtificial Sequence Completely Synthesized Nucleic Acid 8 gggaggacgaugcgggugga guggggauca uacggcuggu agcacgagcu cccuaacagc 60 ggucagacgacucgcccga 79 9 80 RNA Artificial Sequence Description of ArtificialSequence Completely Synthesized Nucleic Acid 9 gggaggacga ugcgggaggaguggagguaa acaauaggcc gguagcgacu cccacuaaca 60 gccucagacg acucgcccga 8010 80 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 10 gggaggacga ugcgguggag uggagguauaccggccggua gcgcauccca cucgggucug 60 ugcucagacg acucgcccga 80 11 80 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 11 gggaggacga ugcgggugga gcggagguuu auacggcugguagcucgagc ucccuaacac 60 gcgguagacg acucgcccga 80 12 80 RNA ArtificialSequence Description of Artificial Sequence Completely SynthesizedNucleic Acid 12 gggaggacga ugcgggugga guggagguau aacggccggu agcgcaucccacucgggucu 60 gcgguagacg acucgcccga 80 13 80 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid13 gggaggacga ugcgggugga guggagggua aacaauggcu gguggcauuc ggaaucuccc 60gcgguagacg acucgcccga 80 14 79 RNA Artificial Sequence Description ofArtificial Sequence Completely Synthesized Nucleic Acid 14 gggaggacgaugcggguugc ugguagccug augugggugg agugagugga ggguugaaaa 60 augcagacgacucgcccga 79 15 79 RNA Artificial Sequence Description of ArtificialSequence Completely Synthesized Nucleic Acid 15 gggaggacga ugcggcugguagcaugugca uugaugggag gaguggaggu caccgucaac 60 cgucagacga cucgcccga 7916 81 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 16 gggaggacga ugcgguuucu cggccaguaguuugcgggug gaguggaggu auaucugcgu 60 ccucgcagac gacucgcccg a 81 17 81 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 17 gggaggacga ugcggcaccu caccuccaua uugccgguuaucgcguaggg ugagcccaga 60 cacgacagac gacucgcccg a 81 18 80 RNA ArtificialSequence Description of Artificial Sequence Completely SynthesizedNucleic Acid 18 gggaggacga ugcggcacuc accuucauau uggccgccau ccccaggguugagcccagac 60 acagcagacg acucgcccga 80 19 81 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid19 gggaggacga ugcgggcaua gugggcaucc caggguugcc uaacggcauc cgggguuguu 60auuggcagac gacucgcccg a 81 20 78 RNA Artificial Sequence Description ofArtificial Sequence Completely Synthesized Nucleic Acid 20 gggaggacgaugcggcagac gacucgcccg aggggauccc ccgggccugc ggaauucgau 60 aucagacgacucgcccga 78 21 62 RNA Artificial Sequence Description of ArtificialSequence Completely Synthesized Nucleic Acid 21 gggaggacga ugcggaacucaaugggccua cuuuuuccgu gguccucaga cgacucgccc 60 ga 62 22 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 22 gggaggacga ugcggaacuc aaugggccua cuuuuccgugguccucagac gacucgcccg 60 a 61 23 60 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 23 gggaggacgaugcggaacuc aaugggccga cuuuuuccgu guccucagac gacucgcccg 60 24 60 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 24 gggaggacga ugcggaacuc aaugggccga cuuuccgugguccucagacg acucgcccga 60 25 61 RNA Artificial Sequence Description ofArtificial Sequence Completely Synthesized Nucleic Acid 25 gggaggacgaugcggaacuc aaugggcnua cuuuuccgug guccucagac gacucgcccg 60 a 61 26 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 26 gggaggacga ugcggaacuc aaugggccga cuuuuccgugguccucagac gacucgcccg 60 a 61 27 60 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 27 gggaggacgaugcggaacuc aaugggccga cuuuuccgug guccucagac gacugcccga 60 28 60 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 28 gggaggacga ugcggacgca ggggaugcuc acuuugacuuuaggccagac gacucgcccg 60 29 60 RNA Artificial Sequence Description ofArtificial Sequence Completely Synthesized Nucleic Acid 29 gggaggacgaugcggacucg gcauucacua acuuuugcgc ucgucagacg acucgcccga 60 30 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 30 gggaggacga ugcggauaac gauucggcau ucacuaacuucucgucagac gacucgcccg 60 a 61 31 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 31 gggaggacgaugcggaugac gauucggcau ucacuaacuu cucgucagac gacucgcccg 60 a 61 32 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 32 gggaggacga ugcggaugac gauucggcau ucacuaacuucucaucagac gacucgcccg 60 a 61 33 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 33 gggaggacgaugcggaugac gauucggcau ucacuaacuu cuacucagac gacucgcccg 60 a 61 34 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 34 gggaggacga ugcggaucug agccuaaagu cauugugaucauccucagac gacucgcccg 60 a 61 35 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 35 gggaggacgaugcgggcguu ggcgauuccu aagugucguu cucgucagac gacucgcccg 60 a 61 36 60 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 36 gggaggacga ugcggcgucu cgagcucuau gcguccucuguggucagacg acucgcccga 60 37 60 RNA Artificial Sequence Description ofArtificial Sequence Completely Synthesized Nucleic Acid 37 gggaggacgaugcggcguca cgagcuuuau gcguucucug uggucagacg acucgcccga 60 38 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 38 gggaggacga ugcggcuuaa aguuguuuau gaucauuccguacgucagac gacucgcccg 60 a 61 39 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 39 gggaggacgaugcgggcguu ggcgauuggu aagugucguu cucgucagac gacucgcccg 60 a 61 40 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 40 gggaggacga ugcgggcguc ucgagcuuua ugcguucucuguggucagac gacucgcccg 60 a 61 41 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 41 gggaggacgaugcgggcguc ucgagcucua ugcguucucu guggucagac gacucgcccg 60 a 61 42 60 RNAArtificial Sequence unsure (52) Description of Artificial SequenceCompletely Synthesized Nucleic Acid. N at position 52 is a or c or g oru 42 ggaggacgau gcggggccua aagucaagug aucauccccu gcgucagacg anucgcccga60 43 61 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 43 gggaggacga ugcggguggc gauuccaagucuuccgugaa cauggucaga cgacucgccc 60 g 61 44 62 RNA Artificial Sequenceunsure (57) Description of Artificial Sequence Completely SynthesizedNucleic Acid. N at position 57 is a or c or g or u 44 gggaggacgaugcgggugac ucgauaucuu ccaaucugua cauggucaga cgacucnccc 60 ga 62 45 61RNA Artificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 45 gggaggacga ugcgguggcg auuccaaguc uuccgugaacauggucagac gacucgcccg 60 a 61 46 58 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 46 gggaggacgaugcgguggcg auuccaaguc uuccgugaac aucagacgac ucgcccga 58 47 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 47 gggaggacga ugcgguccgg cgcgcugagu gccgguuauccucgucagac gacucgcccg 60 a 61 48 62 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 48 gggaggacgaugcgguccgg cgcgcugagu gccgguuuau ccucgucaga cgacucgccc 60 ga 62 49 62RNA Artificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 49 gggaggacga ugcggucuca ugcgccgagu gugaguuuaccuucgucaga cgacucgccc 60 ga 62 50 62 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 50 gggaggacgaugcggucuca ugcgucgagu gugaguuuaa cugcgucaga cgacucgccc 60 ga 62 51 62RNA Artificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 51 gggaggacga ugcggucuca ugcgucgagu gugaguuuaccuucgucaga cgacucgccc 60 ga 62 52 62 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 52 gggaggacgaugcggucugc uacgcugagu ggcuguuuac cuucgucaga cgacucgccc 60 ga 62 53 62RNA Artificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 53 gggaggacga ugcggucgga ugcgccgagu cuccguuuaccuucgucaga cgacucgccc 60 ga 62 54 64 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 54 gggaggacgaugcggugagc gcguauagcg guuucgauag agcugcguca gacgacucgc 60 ccga 64 55 61RNA Artificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 55 gggaggacga ugcggugagc gcguauagcg guuucgauagagccucagac gacucgcccg 60 a 61 56 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 56 gggaggacgaugcggugagc guggcaaacg guuucgauag agccucagac gacucgcccg 60 a 61 57 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 57 gggaggacga ugcggugagc guguaaaacg guuucgauagagccucagac gacucgcccg 60 a 61 58 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 58 gggaggacgaugcggugagc guguaaaacg guuucgauag agccucagac gacucgcccg 60 a 61 59 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 59 gggaggacga ugcggugggc gucagcauuu cgaucuucggcaccucagac gacucgcccg 60 a 61 60 62 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 60 gggaggacgaugcgggaguu guucggcauu uagaucuccg cucccucaga cgacucgccc 60 ga 62 61 62RNA Artificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 61 gggaggacga ugcgggcaaa guucggcauu cagaucuccaugcccucaga cgacucgccc 60 ga 62 62 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 62 gggaggacgaugcggggcuu cucacauauu cuucucuuuc cccgucagac gacucgcccg 60 a 61 63 49 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 63 gggaggagga ucgguguuca gcauucagau cuucagacgacucgcccga 49 64 61 RNA Artificial Sequence unsure (30) Description ofArtificial Sequence Completely Synthesized Nucleic Acid. N at position30 is a or c or g or u 64 gggaggacga ugcgguguuc agcauucagn aucuucacgugucgucagac gacucgcccg 60 a 61 65 60 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 65 gggaggacgaugcgguguuc accauucaga ucuucacgug ucgucagacg acucgcccga 60 66 57 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 66 gggaggacga ugcuguucag cauucagauc uucacgugugucagacgacu cgcccga 57 67 61 RNA Artificial Sequence Description ofArtificial Sequence Completely Synthesized Nucleic Acid 67 gggaggacgaugcgguuucg auagagacuu acaguugagc gcggucagac gacucgcccg 60 a 61 68 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 68 gggaggacga ugcgguuugu gauuuggaag ugggggggauagggucagac gacucgcccg 60 a 61 69 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 69 gggaggacgaugcggugagc guggcaaacg guuucgauag agccucagac gacucgcccg 60 a 61 70 59 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 70 ggagggcgau ggggugagcg uguaaaaggu ugcgauagagccucagacga cucgcccga 59 71 61 RNA Artificial Sequence Description ofArtificial Sequence Completely Synthesized Nucleic Acid 71 gggaggacgaugcggguauc uuaucuuguu uucguuuuuc ugcccucaga cgaucgcccg 60 a 61 72 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 72 gggaggacga ugcggagggu ucuuuucauc uucuuucuuuccccucagac gacucgcccg 60 a 61 73 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 73 gggaggacgaugcggacgaa gaagguggug gaggaguuuc gugcucagac gacucgcccg 60 a 61 74 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 74 gggaggacga ugcggacgaa gaagggggug gaggaguuucgugcucagac gacucgcccg 60 a 61 75 42 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 75 gacgaugcggucucaugcgu cgagugugag uuuaccuucg uc 42 76 62 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid76 gggaggacga ugcggcgauu acugggacgg acucgcgaug ugagcccaga cgacucgccc 60ga 62 77 62 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 77 gggaggacga ugcggcgauu acugggacagacucgcgaug ugagcucaga cgacucgccc 60 ga 62 78 61 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid78 gggaggacga ugcggcgacu acugggaagg gucgcgaagu gagcccagac gacucgcccg 60a 61 79 62 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 79 gggaggacga ugcggcgauu acugggacagacucgcgaug ugagcucaga cgacucgccc 60 ga 62 80 61 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid80 gggaggacga ugcggcgacu acugggagag uacgcgaugu gugcccagac gacucgcccg 60a 61 81 62 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 81 gggaggacga ugcggguccu cggggaaaauuucgcgacgu gaaccucaga cgacucgccc 60 ga 62 82 70 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid82 gggaggacga ugcggcuucu gaagauuauu ucgcgaugug aacuucagac cccucagacg 60acucgcccga 70 83 70 RNA Artificial Sequence Description of ArtificialSequence Completely Synthesized Nucleic Acid 83 gggaggacga ugcggcuucugaagauuauu ucgcgaugug aacuccagac cccucagacg 60 acucgcccga 70 84 62 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 84 gggaggacga ugcggaaagu ggaagugaau ggccgacuugucuggucaga cgacucgccc 60 ga 62 85 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 85 gggaggacgaugcggaaacc aaaucgucga ucuuuccacc gucgucagac gacucgcccg 60 a 61 86 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 86 gggaggacga ugcggaacac gaaacggagg uugacucgaucuggccagac gacucgcccg 60 a 61 87 60 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 87 ggaggacgaugcggaacacg gaagacagug cgacucgauc uggucagacg acucgcccga 60 88 59 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 88 gggaggacga ugcggaacaa ggacaaaagu gcgauucugucuggcagacg acucgcccg 59 89 61 RNA Artificial Sequence Description ofArtificial Sequence Completely Synthesized Nucleic Acid 89 gggaggacgaugcggaacag acgacucgcg caacuacucu gacgucagac gacucgcccg 60 a 61 90 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 90 gggaggacga ugcggaacag guaguugggu gacucugugugaccucagac gacucgcccg 60 a 61 91 60 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 91 ggaggacgaugcggaaccaa aucgucgauc uuuccaccgc ucgucagacg acucgcccga 60 92 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 92 gggaggacga ugcggaaccg cuauugaaug ucacugcuucgugcucagac gacucgcccg 60 a 61 93 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 93 gggaggacgaugcggaaccc aaucgucuaa uucgcugcuc aucgucagac gacucgcccg 60 a 61 94 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 94 gggaggacga ugcggaaccc aaucgucuaa uucgcugcucaucgucagac gacucgcccg 60 a 61 95 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 95 gggaggacgaugcggaacuc aaugggccua cuuuuccgug guccucagac gacucgcccg 60 a 61 96 66 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 96 gggaggacga ugcggaagcg gugagucgug gcuuucuccucgauccucgu cagacgacuc 60 gcccga 66 97 61 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid97 gggaggacga ugcggaagga ugacgaggug guugggguuu gugcucagac gacucgcccg 60a 61 98 61 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 98 gggaggacga ugcggacaag acgagaacggggggagcuac cuggccagac gacucgcccg 60 a 61 99 61 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid99 gggaggacga ugcggagaca cuaaacaaau uggcgaccug accgucagac gacucgcccg 60a 61 100 69 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 100 gggaggacga ugcggagagg cucagacgacucgcccgacc acggaugcga ccucagacga 60 cucgcccga 69 101 59 RNA ArtificialSequence Description of Artificial Sequence Completely SynthesizedNucleic Acid 101 gggaggacga ugcggagaug gauggaagug cuagucuucu ggggucagacgacucgccc 59 102 62 RNA Artificial Sequence Description of ArtificialSequence Completely Synthesized Nucleic Acid 102 gggaggacga ugcggagauggauggaagug cuagucuuuc uggggucaga cgacucgccc 60 ga 62 103 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 103 gggaggacga ugcggagcag uugaaagacg ugcguuucguuuggucagac gacucgcccg 60 a 61 104 67 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 104gggaggacga ugcggagcac aauuuuuucc uuuucuuuuc guccacgugc ucagacgacu 60cgcccga 67 105 58 RNA Artificial Sequence Description of ArtificialSequence Completely Synthesized Nucleic Acid 105 gggaggacga ugcggagcugaugaagauga ucucugaccc cucagacgac ucgcccga 58 106 61 RNA ArtificialSequence Description of Artificial Sequence Completely SynthesizedNucleic Acid 106 gggaggacga ugcggagcug aaagcgaagu gcgagguguu ugguccagacgacucgcccg 60 a 61 107 60 RNA Artificial Sequence Description ofArtificial Sequence Completely Synthesized Nucleic Acid 107 ggaggacgaugcggagcgaa agugcgagug auugaccagg ugcucagacg acucgcccga 60 108 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 108 gggaggacga ugcggagcgu gagaacaguu gcgagauugccuggucagac gacucgcccg 60 a 61 109 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 109gggaggacga ugcggaggag agugugguga gggucguuug agggucagac gacucgcccg 60 a61 110 61 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 110 gggaggacga ugcggaggag cugaugaagaugaucucuga ccccucagac gacucgcccg 60 a 61 111 61 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid111 gggaggacga ugcggaguuc ccagccgccu ugauuucucc guggucagac gacucgcccg 60a 61 112 61 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 112 gggaggacga ugcggauaag ugcgaguguaugaggugcgu guggucagac gacucgcccg 60 a 61 113 61 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid113 gggaggacga ugcggaucug aggagcucuu cgucgugcug agggucagac gacucgcccg 60a 61 114 61 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 114 gggaggacga ugcggauccg aaucuuccuuacacguccug cucgucagac gacucgcccg 60 a 61 115 60 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid115 ggaggacgau gcggauccgc aaaccgacag cucgaguucc gccucagacg acucgcccga 60116 61 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 116 gggaggacga ugcggauggu acuuuagucuuccuugauuc cgccucagac gacucgcccg 60 a 61 117 60 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid117 ggaggacgau gcggaugaug acugaacgug cgacucgacc uggccagacg acucgcccga 60118 60 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 118 ggaggacgau gcggaugagg aggaagagucugaggugcug gggucagacg acucgcccga 60 119 61 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid119 gggaggacga ugcggauuuc ggucgacuaa auaggggugg cucgucagac gacucgcccg 60a 61 120 68 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 120 gggaggacga ugcggcaaga ggucagacgacugccccgag uccucccccg gucagacgac 60 ucgcccga 68 121 60 RNA ArtificialSequence Description of Artificial Sequence Completely SynthesizedNucleic Acid 121 gggaggacga ugcggcagug aaaggcgagu uuucuccucu cccucagacgacucgcccga 60 122 61 RNA Artificial Sequence Description of ArtificialSequence Completely Synthesized Nucleic Acid 122 gggaggacga ugcggcaucguucaggagaa uccacuucgc cucgucagac gacucgcccg 60 a 61 123 61 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 123 gggaggacga ugcggcaucu uccuuguucu uccaaccgugcuccucagac gacucgcccg 60 a 61 124 61 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 124gggaggacga ugcggcaucg uaaacaauuu guuccaucuc cgccucagac gacucgcccg 60 a61 125 61 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 125 gggaggacga ugcggcauug uccaaguuuagcuguccgug cucgucagac gacucgcccg 60 a 61 126 60 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid126 gggaggacga ugcggcauag uccggauacu agucaccagc cucguagacg acucgcccga 60127 61 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 127 gggaggacga ugcggccguc ucgauccuucuaugccuucg cucgucagac gacucgcccg 60 a 61 128 65 RNA Artificial Sequenceunsure (34) Description of Artificial Sequence Completely SynthesizedNucleic Acid. N at position 34 is a or c or g or u 128 gggaggacgaugcggcggga aguuugaggu guanuaccug uugucugguc agacgacucg 60 cccga 65 12967 RNA Artificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 129 gggaggacga ugcggcucaa cucucccaca gacgacucgcccgggccucc ucagacgacu 60 cgcccga 67 130 58 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid130 gggaggacga ugcgggacuc cucgaccgac ucgaccggcu cgucagacga cucgccga 58131 61 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 131 ggaggacgau gcgggaacca aaucgucgaucuuuccaccg cucgucagac gacucgcccg 60 a 61 132 61 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid132 gggaggacga ugcgggacca ccucgauccu cagcgccauu gcccucagac gacucgcccg 60a 61 133 57 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 133 gggaggacga ugcgggaagu ggaaggguaguugugugacc ucagacgacu cgcccga 57 134 67 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid134 cggaggacga ugcgggcaaa cuuuuccuuu ucccuuuauc uuccuugccc ucagacgacu 60cgcccga 67 135 61 RNA Artificial Sequence Description of ArtificialSequence Completely Synthesized Nucleic Acid 135 gggaggacga ugcggggccgacgauucacc aauguucucu cuggucagac gacucgcccg 60 a 61 136 62 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 136 ggaggacgau gcgggguucc ucaaugacga ucuccauuccgcucgucaga cgacucgccc 60 ag 62 137 61 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid137 ggaggacgau gcgggucgac auugaagcug cucugccuug auccucagac gacucgcccg 60a 61 138 61 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 138 gggaggacga ugcgguccaa uucguucucaugccuuuccg cucgucagac gacucgcccg 60 a 61 139 59 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid139 gggaggacga ugcgguccgc aaguuuagca cucacugccu cgucagacga cucgcccga 59140 58 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 140 gggaggacga ugcgguccac aucgaauuuucuguccguuc gucagacgac ucgcccga 58 141 63 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid141 gggaggacga ugcggucgau guucuuccuc accacugcuc gucgccucag acgacucgcc 60cga 63 142 62 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 142 gggaggacga ugcggucgag cugagaggggcuacuuguuc uggucacaga cgacucgccc 60 ga 62 143 63 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid143 gggaggacga ugcgguggaa gcgaaugggc uagggugggc ugaccuccag acgacucgcc 60cga 63 144 64 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 144 gggaggacga ugcgguggac uucuuuuccucuuccuccuu ccgccgguca gacgacucgc 60 ccga 64 145 60 RNA ArtificialSequence Description of Artificial Sequence Completely SynthesizedNucleic Acid 145 ggaggacgau gcgguuccaa aucgucuaag caucgcucgc ucgucagacgacucgcccag 60 146 62 RNA Artificial Sequence Description of ArtificialSequence Completely Synthesized Nucleic Acid 146 gggaggacga ugcgguuccacaucgcaauu uucuguccgu gcucgucaga cgacucgccc 60 ga 62 147 60 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 147 gggaggacga ugcgguucca caucgaauuu ucuguccgugucgucagacg acucgcccga 60 148 61 RNA Artificial Sequence Description ofArtificial Sequence Completely Synthesized Nucleic Acid 148 gggaggacgaugcgguuccg aucgacucca cauacaucug cucgucagac gacucgcccg 60 a 61 149 61RNA Artificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 149 gggaggacga ugcgguuccg acaucgaugu ugcucuucgccucgucagac gacucgcccg 60 a 61 150 63 RNA Artificial Sequence Descriptionof Artificial Sequence Completely Synthesized Nucleic Acid 150gggaggacga ugcgguuccg aaguucuucc cccgagccuu cccccuccag acgacucgcc 60 cga63 151 61 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 151 gggaggacga ugcgguuccg acgauucuccaauguucucu cuggucagac gacucgcccg 60 a 61 152 61 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid152 gggaggacga ugcgguuccg acgauucucc aaucuucucu cuggucagac gacucgcccg 60a 61 153 61 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 153 gggaggacga ugcgguuccg caaguuuagacacucacugc cucgucagac gacucgcccg 60 a 61 154 61 RNA Artificial Sequenceunsure (33) Description of Artificial Sequence Completely SynthesizedNucleic Acid. N at position 33 is a or c or g or u 154 gggaggacgaugcgguuccg caaaguagau aunucauccg cacgucagac gacucgcccg 60 a 61 155 61RNA Artificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 155 gggaggacga ugcgguugag uggacagugc gauucguuuuggggucagac gacucgcccg 60 a 61 156 98 DNA Artificial Sequence unsure(33)..(72) Description of Artificial Sequence Completely SynthesizedNucleic Acid. N′s at positions 33-72 are a or c or g or t. 156taatacgact cactataggg aggacgatgc ggnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn nnnnnnnnnn nncagacgac tcgcccga 98 157 81 RNA ArtificialSequence unsure (16)..(65) Description of Artificial Sequence CompletelySynthesized Nucleic Acid. N′s at positions 16-65 are a or c or g or u.157 gggaggacga ugcggnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnncagac gacucgcccg a 81 158 40 DNA Artificial Sequence Description ofArtificial Sequence Completely Synthesized Nucleic Acid 158 taatacgactcactataggg agataagaat aaacgctcaa 40 159 24 DNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid159 gcctgttgtg agcctcctgt cgaa 24 160 38 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid160 cgaugcgguc ucaugcgucg agugugaguu uaccuucg 38 161 36 RNA ArtificialSequence Description of Artificial Sequence Completely SynthesizedNucleic Acid 161 gaugcggucu caugcgucga gugugaguuu accuuc 36 162 34 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 162 augcggucuc augcgucgag ugugaguuua ccuu 34163 90 DNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 163 gggagataag aataaacgct caaggacgatgcggtctcat gcgtcgagtg tgagtttacc 60 ttcgtcttcg acaggaggct cacaacaggc 90164 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 164 gggagauaag aauaaacgcu caagugcgacgcggucucga gcgcggaguu cgaguuuacc 60 uucgcauucg acaggaggcu cacaacaggc 90165 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 165 gggagauaag aauaaacgcu caagcucgacgcggucccag gcguggaguc uggguuuacc 60 uucgaguucg acaggaggcu cacaacaggc 90166 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 166 gggagauaag aauaaacgcu caagaaccacgcggucucag gcguagaguc ugaguuuacc 60 uugguuuucg acaggaggcu cacaacaggc 90167 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 167 gggagauaag aauaaacgcu caagaaccacgcggucucag gcguagaguc uguguuuacc 60 uugguuuucg acaggaggcu cacaacaggc 90168 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 168 gggagauaag aauaaacgcu caagugcgacgcggucucga gcgcggaguu cgaguucacc 60 uucgcauucg acaggaggcu cacaacaggc 90169 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 169 gggagauaag aauaaacgcu caagcacaacgcggucucau gcgucgagua ugaguuuacc 60 uuuguguucg acaggaggcu cacaacaggc 90170 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 170 gggagauaag aauaaacgcu caagguccucgcggucucau gcgccgagua ugaguuuacc 60 uaggacuucg acaggaggcu cacaacaggc 90171 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 171 gggagauaag aauaaacgcu caaggucgucgcggucugau gcgcugagua ucaguuuacc 60 uacgacuucg acaggaggcu cacaacaggc 90172 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 172 gggagauaag aauaaacgcu caagguacacgcggucugac gcgcugagug ucaguuuacc 60 uuguacuucg acaggaggcu cacaacaggc 90173 91 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 173 gggagauaag aauaaacgcu caagaaaccacgcggucuca ggcgcagagu cugaguuuac 60 cuucgcauuc gacaggaggc ucacaacagg c91 174 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 174 gggagauaag aauaaacgcu caagaaccacgcggucucag gcgcagaguc ugaguuuacc 60 uugguuuucg acaggaggcu cacaacaggc 90175 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 175 gggagauaag aauaaacgcu caaggacgccgcggucucag gcgcugaguc ugaguuuacc 60 ugcgucuucg acaggaggcu cacaacaggc 90176 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 176 gggagauaag aauaaacgcu caaggcugacgcggucucag gcguggaguc ugaguuuacc 60 uucggcuucg acaggaggcu cacaacaggc 90177 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 177 gggagauaag aauaaacgcu caagcaugacgcggucucag gcguggaguc ugaguuuacc 60 uucguguucg acaggaggcu cacaacaggc 90178 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 178 gggagauaag aauaaacgcu caaggucgacgcggucucag gcguugaguc uguguuuacc 60 uucgacuucg acaggaggcu cacaacaggc 90179 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 179 gggagauaag aauaaacgcu caaggucgacgcggucucag gcguugaguc uguguuuacc 60 uucgacuucg acaggaggcu cacaacaggc 90180 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 180 gggagauaag aauaaacgcu caaggacgccgcggucucag gcguugaguc ugaguuuacc 60 ugcgucuucg acaggaggcu cacaacaggc 90181 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 181 gggagauaag aauaaacgcu caaggacgacgcggucugau gcgcugagug ucaguuuacc 60 uucgucuucg acaggaggcu cacaacaggc 90182 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 182 gggagauaag aauaaacgcu caagaacgacgcggucugau gcgcugagug ucaguguacc 60 uucgucuucg acaggaggcu cacaacaggc 90183 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 183 gggagauaag aauaaacgcu caaggucgacgcggucugau gcguagagug ucaguuuacc 60 uucgacuucg acaggaggcu cacaacaggc 90184 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 184 gggagauaag aauaaacgcu caaggucgacgcggucugau gcguagagug ucaguucacc 60 uucgacuucg acaggaggcu cacaacaggc 90185 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 185 gggagauaag aauaaacgcu caaguacgacgcggucccgu gcguggagug cggguuuacc 60 uucguauucg acaggaggcu cacaacaggc 90186 90 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 186 gggagauaag aauaaacgcu caaggacgacgcggucugau gcgcagagug ucgguuuacc 60 uuugucuucg acaggaggcu cacaacaggc 90187 90 RNA Artificial Sequence unsure (33) Description of ArtificialSequence Completely Synthesized Nucleic Acid. N at position 33 is a or cor g or u 187 gggagauaag aauaaacgcu caaggacgac gcngucugau gcgcagagugucaguuuacc 60 uucgacuucg acaggaggcu cacaacaggc 90 188 90 RNA ArtificialSequence Description of Artificial Sequence Completely SynthesizedNucleic Acid 188 gggagauaag aauaaacgcu caaggacgac gcggucugau gcgcagagugucaguuuacc 60 uucgucuucg acaggaggcu cacaacaggc 90 189 90 RNA ArtificialSequence Description of Artificial Sequence Completely SynthesizedNucleic Acid 189 gggagauaag aauaaacgcu caaggacgac gcggucggau gcgcagaguguccguuuacc 60 uucgucuucg acaggaggcu cacaacaggc 90 190 38 RNA ArtificialSequence Description of Artificial Sequence Completely SynthesizedNucleic Acid 190 cgccgcgguc ucaggcgcug agucugaguu uaccugcg 38 191 37 RNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 191 cgccgcgguu cgggcgcuga gucugaguuu accugcg 37192 38 RNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 192 cgccgcgguc ucaggcgcug agucugaguuuaccugcg 38 193 38 RNA Artificial Sequence Description of ArtificialSequence Completely Synthesized Nucleic Acid 193 cgaugcgguc ucaugcgucgagugugaguu uaccuucg 38 194 38 RNA Artificial Sequence Description ofArtificial Sequence Completely Synthesized Nucleic Acid 194 cgccgcggucucaggcgcug agucugaguu uaccugcg 38 195 38 RNA Artificial SequenceDescription of Artificial Sequence Completely Synthesized Nucleic Acid195 cgccgcgguc ucaggcgcug agucugaguu uaccugcg 38 196 38 RNA ArtificialSequence Description of Artificial Sequence Completely SynthesizedNucleic Acid 196 cgccgcgguc ucaggcgcug agucugaguu uaccugcg 38 197 37 DNAArtificial Sequence Description of Artificial Sequence CompletelySynthesized Nucleic Acid 197 ggcggggcta cgtaccgggg ctttgtaaaa ccccgcc 37198 25 DNA Artificial Sequence Description of Artificial SequenceCompletely Synthesized Nucleic Acid 198 ctctcgcacc catctctctc cttct 25

We claim:
 1. A method for treating a Complement System-mediated diseasecomprising administering to a patient in need thereof a pharmaceuticallyeffective amount of a Nucleic Acid Ligand of a Complement SystemProtein.
 2. The method of claim 1 wherein said Nucleic Acid Ligand isidentified according to a method comprising: a) preparing a candidatemixture of nucleic acids; b) contacting the candidate mixture of nucleicacids with a Complement System Protein, wherein nucleic acids having anincreased affinity to said Complement System Protein relative to thecandidate mixture may be partitioned from the remainder of the candidatemixture; c) partitioning the increased affinity nucleic acids from theremainder of the candidate mixture; and d) amplifying the increasedaffinity nucleic acids to yield a mixture of nucleic acids enriched fornucleic acid sequences with relatively higher affinity and specificityfor binding said Complement System Protein, wherein Nucleic Acid Ligandsof said Complement System Protein may be identified.
 3. The method ofclaim 1 wherein said Complement System Protein is selected from thegroup consisting of C1q, C3 and C5.
 4. The method of claim 1 whereinsaid Complement System-mediated disease is selected from the groupconsisting of myocardial infarction and Alzheimer's disease.
 5. A methodof treating a Complement System-mediated disease comprisingadministering to a patient in need thereof a pharmaceutical agent whichspecifically treats said disease and administering a Nucleic Acid LigandC1q inhibitor in an amount effective to inhibit activation of theComplement System.